Generation of aheterozygous knockout pig model of arrhythmogenic cardiomyopathy.

Abstract

Arrhythmogenic cardiomyopathy (ACM) confers elevated risk of ventricular arrhythmias and sudden cardiac death, yet limitations in early lesion sampling and model development continue to hinder mechanistic and translational research. Clinical, pathological, and mutational profiles were examined in 24 individuals with ACM harboringvariants. Among these, a patient carrying the c.1132C>T mutation exhibited the earliest onset and presented both structural cardiac abnormalities and major adverse cardiovascular events. To facilitate disease modeling, the c.1147C>T variant-a previously reported pathogenic substitution located proximal to position c.1132 in-was selected to enhance the feasibility of generating a porcine model. The BE3 gene editing system was used to induce C>T mutation. Two single guide RNAs targeting thegene were designed (sgRNA1 for c.1132C>T and sgRNA2 for c.1147C>T), yielding editing efficiencies of 42.9% and 25.9%, respectively. SgRNA1 was used to generateporcine fetal fibroblasts. A total of 14 cloned piglets were produced, including 11 viable and three stillbornindividuals. By 24 months of age,pigs developed premature ventricular contractions and right ventricular dilatation. Histological analysis revealed adipocyte infiltration within the right ventricular wall, and electron microscopy demonstrated reduced desmosomal length and electron density consistent with desmosomal dysfunction. Transcriptomic profiling showed high expression of genes associated with lipid catabolic processes. This study established the firstporcine model of ACM using BE3-mediated base editing, providing a valuable platform for elucidating early pathogenic mechanisms and evaluating therapeutic interventions.