Human c-MYBPC3 RNA Targeted Therapy, Reversal of Hypertrophic Cardiomyopathy in the Zebrafish Model

Abstract

Hypertrophic cardiomyopathy (HCM) is a serious heart disease and is defined as abnormal left ventricular (LV) wall thickening with diastolic dysfunction. HCM is an autosomal dominant monogenic disease caused by a mutation in 1 of 13 or more genes encoding protein components of sarcomere (i.e. sarcomere is the subunit for muscle tissue). The myosin binding protein C (MYBPC) encoded by mybpc3 gene, a key constituent of the thick filaments of the sarcomere (Dhandapany et al., 2009). By binding to myosin, titin, and actin, MYBPC contributes to maintaining the structural integrity of the sarcomere and regulates cardiac contractility and relaxation (Harris et al., 2002). Mutations of c-MYBPC3 gene have been demonstrated to be associated with a risk of cardiac hypertrophy and represent one of the common causes of HCM with about more than 20% frequency (Houston & Stevens, 2015). Zebrafish is a widely used animal model for the cardiac genotype – phenotype association since it allows easy genetic manipulation. We have previously identified four disease causing missense mutations of MYBPC3 domain C1 in cardiac patients: Mutation1 (Arg177His), Mutation 2 (Ala216Thr), Mutation 3 (Glu258Lys) and Mutation 4 (Ser217Gly). Previously, it was shown that mybpc3 gene mutations induced a zebrafish embryonic phenotype resembling HCM(Chen et al., 2013). We have recapitulated these mutations in the zebrafish model (Da'as et al., 2014). The efficacy of human RNA injection to zebrafish embryos for rescuing the induced hypertrophic defects was recently suggested as a novel rescue strategy for HCM (Behrens-Gawlik, Mearini, Gedicke-Hornung, Richard, & Carrier, 2014). Previously, we showed that, zebrafish specific cardiac phenotypes resembling the different human mybpc3 mutations were partially reverted upon co-injection of Human c-MYBPC3 mRNA (Da'as et al, 2015). In the current study, we induced hypertrophic condition to zebrafish embryos with morpholino injections to target exon 5 (Mutation 1, 2 and 4) and exon 6 (Mutation 3). We have also analyzed the recovery of these conditions with RNA co-injection.