Mechanistic Insights Into RNA Splicing As A Trigger For Arrhythmogenic Right Ventricular Cardiomyopathy

Abstract

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disease that causes sudden death in young adults and athletes. ARVC is termed a “disease of the desmosome” as 40% of mutations in ARVC patients are found in the cardiac desmosomal components, with plakophilin‐2 (PKP2) being the most frequently mutated desmosomal gene in ARVC. Evidence from humans suggests that altered RNA splicing may be a critical mechanism through which PKP2 patient genetics drive ARVC. Studies have suggested that approximately one third of all human disease‐causing mutations are a result of defects in RNA splicing; highlighting the importance of testing the relevance of this mechanism in human ARVC. However, there are no models and limited mechanistic insights into how mutations in RNA splicing impact ARVC disease pathogenesis. We hypothesize that RNA splicing mutations in PKP2 are sufficient to recapitulate classic disease features associated with human ARVC and can provide valuable mechanistic insight into how altered RNA splicing impacts disease. Through CRISPR‐Cas9 we generated a novel knock‐in mouse model harboring a human equivalent PKP2 mutation (IVS10‐1 G>C) that impacts RNA splicing and is sufficient to recapitulate all classic ARVC disease features. PKP2 homozygous mutant (PKP2 Hom) mice are viable at birth yet display adult hallmarks of ARVC including ventricular arrhythmias, right and left ventricular dysfunction, and fibro‐fatty replacement of myocardium leading to sudden death. RNA and sequencing analyses of exons spanning the PKP2 mutation site reveals a larger PKP2 transcript that retains a 54 base pair portion of the intronic sequence in PKP2 Hom hearts. However, RNA analysis of exons outside the PKP2 mutation site reveals PKP2 RNA transcripts at similar levels to wild type PKP2, suggesting that total RNA levels are not impacted by the mutation. Instead, we show that PKP2 Hom hearts express a higher molecular weight mutant PKP2 protein in the absence of endogenous PKP2. Analysis of PKP2 Hom neonatal hearts and cardiomyocytes reveal early desmosomal protein loss coupled with a susceptibility to baseline arrhythmias in the absence of overt cardiac disease features at this stage (e.g., no changes in heart weight/body weight ratios, no upregulation of cardiac stress and fibrotic markers). We provide a novel mouse model that highlights the sufficiency and molecular consequences of a PKP2 RNA splicing mutation that triggers all of the classic early and adult onset disease features associated with human ARVC.Support or Funding InformationNational Science Foundation (Graduate Research Fellowship Program), National Institutes of Health (NIH R01, HL142251‐A1)