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

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). 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

CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations

“…To select gRNAs for our experiments, we pre-screened 23 gRNAs with high in vivo on-target efficiency. These gRNAs target zebrafish orthologues of human genes in loci identified by genome-wide association studies (GWAS) for cardiometabolic risk factors and diseases 34 – 36 and have been used to examine the role of the candidate genes in cardiometabolic disorders 37 , 38 . We first used genome-wide Nano-OTS 33 to identify off-target Cas9 cleavage activity in vitro for all 23 gRNAs (Supplementary Tables S1 , S2 ).…”

“…In this study, we used long-read sequencing to examine on- and off-target genome editing outcomes, across multiple stages of development and across generations. Genome editing was accomplished using standard routines for germline editing at the single-cell stage, using gRNAs that have recently been used for functional studies of cardiometabolic diseases in zebrafish model systems 37 , 38 . This revealed insertions and deletions of sizes up to several kilobases, at on- and off-target sites, with a high degree of individual-level variation in genome editing outcomes both in the F0 and F1 generations.…”

Drivers of Sinoatrial Node Automaticity in Zebrafish: Comparison With Mechanisms of Mammalian Pacemaker Function

“…It has been shown that compared to other regions of the heart, the zebrafish SAN overexpresses several signature mammalian pacemaker genes, including those encoding HCN and Ca 2+ - and K + -gated channels ( von der Heyde et al, 2020 ; Minhas et al, 2021 ). In the context of SAN function, it has also been shown that knock down of the large-conductance Ca 2+ -activated K + channel (K Ca 1.1) in zebrafish, which is strongly expressed in the human SAN, results in sinus bradycardia ( Pineda et al, 2021 ), so it may play an important role in zebrafish SAN automaticity through its contribution to repolarisation.…”

In vivo identification and validation of novel potential predictors for human cardiovascular diseases

“…F0 mutagenesis screens are becoming more and more relevant [ 21 , 25 , 72 , 73 ], largely due to the improvements in CRISPR/Cas9 gene targeting efficiency by modification of the enzyme or promoting nuclear localization [ 31 ]. Overall, gene targeting in medaka using CRISPR/Cas9 has proven to be highly efficient, as shown by the prominent loss of GFP expression in gfp crispants when injected at the 1-cell stage ( Fig 1B and S1 Fig ) and the prominent bi-allelic mutagenesis as apparent by the loss of eye-pigmentation in the oca2 crispants ( Fig 1C and S2A Fig ).…”