Translating GWAS-identified loci for cardiac rhythm and rate using an in vivo image- and CRISPR/Cas9-based approach

Benedikt Von Der Heyde,Harold Snieder,Eco De Geus,Olga Dethlefsen,Anastasia Emmanouilidou,Tiffany Klingström,Erik Ingelsson,Eugenia Mazzaferro,Hannah L Brooke,Marcel Den Hoed,Ida Höijer,Sitaf Jumaa,Silvia Vicenzi,Adam Ameur,Amin Allalou

doi:10.1038/s41598-020-68567-1

Abstract

A meta-analysis of genome-wide association studies (GWAS) identified eight loci that are associated with heart rate variability (HRV), but candidate genes in these loci remain uncharacterized. We developed an image- and CRISPR/Cas9-based pipeline to systematically characterize candidate genes for HRV in live zebrafish embryos. Nine zebrafish orthologues of six human candidate genes were targeted simultaneously in eggs from fish that transgenically express GFP on smooth muscle cells (Tg[acta2:GFP]), to visualize the beating heart. An automated analysis of repeated 30 s recordings of beating atria in 381 live, intact zebrafish embryos at 2 and 5 days post-fertilization highlighted genes that influence HRV (hcn4 and si:dkey-65j6.2 [KIAA1755]); heart rate (rgs6 and hcn4); and the risk of sinoatrial pauses and arrests (hcn4). Exposure to 10 or 25 µM ivabradine—an open channel blocker of HCNs—for 24 h resulted in a dose-dependent higher HRV and lower heart rate at 5 days post-fertilization. Hence, our screen confirmed the role of established genes for heart rate and rhythm (RGS6 and HCN4); showed that ivabradine reduces heart rate and increases HRV in zebrafish embryos, as it does in humans; and highlighted a novel gene that plays a role in HRV (KIAA1755).

Highlights

Heart rate variability (HRV) reflects the inter-beat variation of the RR interval
A trend for an effect on sinoatrial pauses was observed for the SYT10 orthologue, albeit with an opposite direction of effect at 2 and 5dpf. We examined whether these four genes are already targeted by existing, FDA-approved medication using the drug-gene interaction (DGI) database
Given the role of mutations in hcn[4] on heart rate variability (HRV) and heart rate, we examined the effect of 24 h of exposure to 0, 10 or 25 μM ivabradine in DMSO on these outcomes at 5dpf, in embryos free from CRISPR/Cas9-induced mutations

Summary

Introduction

Heart rate variability (HRV) reflects the inter-beat variation of the RR interval. HRV is controlled by the sinoatrial node, which receives input from the autonomic nervous system. Rodents are not suitable for high-throughput, in vivo characterization of cardiac rhythm and rate Such screens are essential to systematically characterize positional candidate genes in the large number of loci that have been identified in GWAS for H RV3, heart rate[6] and cardiac conduction[7,8]. The zebrafish has a wellannotated genome, with orthologues of at least 71.4% of human g enes[19] These genes can be targeted efficiently and in a multiplexed manner using Clustered, Regulatory Interspaced, Short Palindromic Repeats (CRISPR) and CRISPR-associated systems (Cas)[20]. All characteristics combined make zebrafish embryos an attractive model system to systematically characterize candidate genes for cardiac rhythm, rate and conduction

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