Reduced cardiac vagal control reflected in low heart rate variability (HRV) is associated with greater risks for cardiac morbidity and mortality. In two-stage meta-analyses of genome-wide association studies for three HRV traits in up to 53,174 individuals of European ancestry, we detect 17 genome-wide significant SNPs in eight loci. HRV SNPs tag non-synonymous SNPs (in NDUFA11 and KIAA1755), expression quantitative trait loci (eQTLs) (influencing GNG11, RGS6 and NEO1), or are located in genes preferentially expressed in the sinoatrial node (GNG11, RGS6 and HCN4). Genetic risk scores account for 0.9 to 2.6% of the HRV variance. Significant genetic correlation is found for HRV with heart rate (−0.74

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). 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. Autonomic imbalance has been associated with work stress and other modifiable or non-modifiable risk factors 1 , and is reflected in lower HRV. Lower HRV has been associated with higher cardiac morbidity and mortality 2 , as well as with a higher risk of all-cause mortality 3. HRV can be quantified non-invasively using an ECG, making HRV a useful clinical marker for perturbations of the autonomic nervous system. However, the mechanisms influencing HRV remain poorly understood. Recently, we and others identified the first loci that are robustly associated with HRV, using a meta-analysis of genome-wide association studies (GWAS) with data from 53,174 participants 3. Five of the identified loci had

31A meta-analysis of genome-wide association studies (GWAS) identified eight loci 32 that are associated with heart rate variability (HRV) in data from 53,174 individuals. 33 However, functional follow-up experiments -aiming to identify and characterize 34 causal genes in these loci -have not yet been performed. We developed an image-and 35 CRISPR-Cas9-based pipeline to systematically characterize candidate genes for HRV 36 in live zebrafish embryos and larvae. Nine zebrafish orthologues of six human 37 candidate genes were targeted simultaneously in fertilized eggs from fish that 38 transgenically express GFP on smooth muscle cells (Tg(acta2:GFP)), to visualize the 39 beating heart using a fluorescence microscope. An automated analysis of repeated 30s 40 recordings of 381 live zebrafish atria at 2 and 5 days post-fertilization highlighted 41 genes that influence HRV (hcn4 and si:dkey-65j6.2); heart rate (rgs6 and hcn4) and 42 the risk of sinoatrial pauses and arrests (hcn4). Hence, our screen confirmed the role 43 of established genes for heart rate and rhythm (rgs6 and hcn4), and highlighted a 44 novel gene implicated in HRV (si:dkey-65j6.2). 45 46 100 101 6