Background: Mutations in the desmoplakin (DSP) gene have been identified in patients affected with Arrhythmogenic Cardiomyopathy type 8 (AC8), a progressive heart muscle disorder, frequently involved in juvenile sudden death due to cardiac arrhythmias. In spite of the recent discovery of genes whose mutations cause ACs, early molecular events leading to cell death and arrhythmias remain elusive. In the present study we evaluate the pathogenic mechanisms of DSP dysfunction in vivo, using zebrafish (Danio rerio) as an innovative and promising model for this life-threatening arrhythmic disorder. Purpose: The aim of the present study is the generation of transient AC8 zebrafish models, using a morpholino-mediated knock-down strategy, and their structural and functional characterization. In addition, by exploiting zebrafish pathway reporter lines, we aim to study cell signaling alterations potentially involved in AC8 pathogenesis. The final goal is the assessment of our zebrafish AC8 models as a suitable tool for pathway-directed drug screening. Methods: A morpholino (MO)-based antisense strategy was used to obtain the knockdown of zebrafish dspa and dspb genes, both orthologous to human DSP. Transient AC8 zebrafish models were morphologically characterized and, subsequently, functionally tested for alterations in Wnt, Bmp, TGFbeta, FGF, Shh, Notch, CREB, Hippo and Hypoxia signaling. Results: The knock-down of both dspa and dspb leads to delayed development, microcephaly, pericardial edema and altered heart rate. Interestingly, TEM analysis of zebrafish tissues under dspa+dspb simultaneous knock-down shows highly reduced and disorganized desmososmes, resembling “pale” desmosomes identified in endomyocardial biopsies from AC patients. Moreover, the analysis of signaling pathways detects a cardiac-specific reduction of Wnt signaling responsiveness in both dspa and dspb knock-down embryos, confirming previous evidences that DSP suppression leads to a reduction of canonical Wnt signaling in cellular and mouse models. Conclusions: Our transient knock-down of zebrafish Dsp genes is able to recapitulate some AC8 features, such as desmosomal defects and heart rate alteration, pointing to zebrafish as a suitable model for the in vivo screening of molecularly-targeted drugs. Moreover, confirmation of the reduction in canonical Wnt signaling due to DSP knock-down suggests that this pathway could be a general mechanism involved in the pathogenesis of desmosomal-associated AC forms, and, thus, a promising target for AC therapeutic intervention.

Desmoplakin knock-downs in zebrafish recapitulate arrhythmogenic cardiomiopathy type 8 and involve Wnt/beta-catenin signaling in AC8 pathogenesis

BEFFAGNA, GIORGIA;GIULIODORI, ALICE;PILICHOU, KALLIOPI;ARGENTON, FRANCESCO;BASSO, CRISTINA;THIENE, GAETANO;TISO, NATASCIA
2017

Abstract

Background: Mutations in the desmoplakin (DSP) gene have been identified in patients affected with Arrhythmogenic Cardiomyopathy type 8 (AC8), a progressive heart muscle disorder, frequently involved in juvenile sudden death due to cardiac arrhythmias. In spite of the recent discovery of genes whose mutations cause ACs, early molecular events leading to cell death and arrhythmias remain elusive. In the present study we evaluate the pathogenic mechanisms of DSP dysfunction in vivo, using zebrafish (Danio rerio) as an innovative and promising model for this life-threatening arrhythmic disorder. Purpose: The aim of the present study is the generation of transient AC8 zebrafish models, using a morpholino-mediated knock-down strategy, and their structural and functional characterization. In addition, by exploiting zebrafish pathway reporter lines, we aim to study cell signaling alterations potentially involved in AC8 pathogenesis. The final goal is the assessment of our zebrafish AC8 models as a suitable tool for pathway-directed drug screening. Methods: A morpholino (MO)-based antisense strategy was used to obtain the knockdown of zebrafish dspa and dspb genes, both orthologous to human DSP. Transient AC8 zebrafish models were morphologically characterized and, subsequently, functionally tested for alterations in Wnt, Bmp, TGFbeta, FGF, Shh, Notch, CREB, Hippo and Hypoxia signaling. Results: The knock-down of both dspa and dspb leads to delayed development, microcephaly, pericardial edema and altered heart rate. Interestingly, TEM analysis of zebrafish tissues under dspa+dspb simultaneous knock-down shows highly reduced and disorganized desmososmes, resembling “pale” desmosomes identified in endomyocardial biopsies from AC patients. Moreover, the analysis of signaling pathways detects a cardiac-specific reduction of Wnt signaling responsiveness in both dspa and dspb knock-down embryos, confirming previous evidences that DSP suppression leads to a reduction of canonical Wnt signaling in cellular and mouse models. Conclusions: Our transient knock-down of zebrafish Dsp genes is able to recapitulate some AC8 features, such as desmosomal defects and heart rate alteration, pointing to zebrafish as a suitable model for the in vivo screening of molecularly-targeted drugs. Moreover, confirmation of the reduction in canonical Wnt signaling due to DSP knock-down suggests that this pathway could be a general mechanism involved in the pathogenesis of desmosomal-associated AC forms, and, thus, a promising target for AC therapeutic intervention.
2017
ECAS 2017
File in questo prodotto:
File Dimensione Formato  
Final_Program_ECAS-2017.pdf

accesso aperto

Descrizione: Congress program
Tipologia: Altro materiale allegato
Licenza: Accesso gratuito
Dimensione 1.92 MB
Formato Adobe PDF
1.92 MB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11577/3228281
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact