Natural genetic variation quantitatively regulates heart rate and dimension

Abstract

The polygenic contribution to heart development and function along the health-disease continuum remains unresolved. To gain insight into the genetic basis of quantitative cardiac phenotypes, we utilize highly inbred Japanese rice fish models, Oryzias latipes, and Oryzias sakaizumii. Employing automated quantification of embryonic heart rates as core metric, we profiled phenotype variability across five inbred strains. We observed maximal phenotypic contrast between individuals of the HO5 and the HdrR strain. HO5 showed elevated heart rates associated with embryonic ventricular hypoplasia and impaired adult cardiac function. This contrast served as the basis for genome-wide mapping. In an F2 segregation population of 1192 HO5 x HdrR embryos, we mapped 59 loci (173 genes) associated with heart rate. Experimental validation of the top 12 candidate genes by gene editing revealed their causal and distinct impact on heart rate, development, ventricle size, and arrhythmia. Our study uncovers new diagnostic and therapeutic targets for developmental and electrophysiological cardiac diseases and provides a novel scalable approach to investigate the intricate genetic architecture of the vertebrate heart.

Fig. 1. Cardiac phenotype contrast in medaka inbred strains.

A Layout of the automated heartbeat detection in medaka embryos in native environment (28 °C) using high-throughput imaging and image-based heart rate quantification; positional effects were excluded; (B) Distribution of embryonic heart rates in five inbred strains derived from Southern Japanese medaka populations (HdrR, HO5, Cab) and Northern Japanese populations (HNI, Kaga) across embryonic development starting with the onset of heartbeat. Heart rates of 6–18 embryos per strain (Table S1) were determined every 4 h under a 12 h-light/12 h-dark cycle; heart rate measurements for each strain plotted as mean heart rates ± SD; dotted lines, window of circadian-rhythm-stable heartbeat for comparative analysis (100–104 h post fertilization, hpf). C Cardiac morphology in HdrR and HO5 hatchlings; end-systolic frame, scale bar, 50 µm (n = 3 for each strain). D, E Exercise assessment and swim performance of adult fish in a swim tunnel assay (movie S1). White line in (E) used for kymograph - note stable swimming behavior in HdrR versus fluctuating HO5 individual. (F) Pulsed-wave (pw) doppler of ventricular inflow (atrium-ventricle) and outflow (ventricle-bulbus arteriosus) tracts. Schemes are adapted from a previous publication under a CC BY 4.0 Creative Commons licence.

Fig. 2. F2 segregation analysis reveals temperature-sensitive QTLs affecting the heartbeat.

A Crossing setup used to generate HdrR × HO5 offspring with segregated SNPs in the second generation: isogenic HO5 and HdrR parents are crossed to generate hybrid (heterozygous) F1 generation (gray) with intermediate phenotype, which after incrossing results in F2 individuals (green) with individually segregated SNPs resulting from one cycle of meiotic recombination. Automated embryonic heartbeat analysis at 4 days post-fertilization (dpf), performed on all 1260 individual embryos and challenged by increasing temperatures (21 °C, 28 °C, 35 °C). Whole-genome sequencing of measured individuals (1192 genomes) with an effective average coverage of 0.78× allows phenotype-genotype correlation. B Individual embryonic heart rates of the inbred strains HdrR (slow heart rate, blue) and HO5 (fast heart rate, orange) increase with temperature (21 °C, 28 °C, and 35 °C). The F2 individuals with recombined HdrR × HO5 genomes (green) span the range of parental (F0) heart rates between the two strains with a subgroup of F2 individuals exhibiting heart rate variance beyond the parental extremes (21 °C and 28 °C); sample sizes (n) for 21 °C, 28 °C and 35 °C: n (HdrR F0) = 35, 35, 35, n (HO5 F0) = 20, 22, 22, n (HdrR × HO5 F2) = 1260, 1260, 1260. C Minus log10 p values from genome-wide association tests of recombination block genotypes and heart rate measures at different temperatures using a linear mixed model. Chromosomes 3 and 5 hold the most segregated recombination blocks associated with heart rate differences. Twelve selected genes from the loci passing the significance threshold are indicated; red line: 1% false discovery rate (FDR), blue line: 5% FDR, determined by permutation. Schemes are adapted from a previous publication under a CC BY 4.0 Creative Commons licence.

Fig. 3. Phenotype proportions in knockout models of cardiac candidate genes.

A Validation workflow encompassing zygotic microinjections using a HdrR (myl7::eGFP; myl7::H2A-mCherry) reporter line, followed by phenotypic classification of embryos with normal developed hearts and embryos with cardiac-specific phenotypes 4 days post-fertilization (dpf). B Proportion (bars) and counts (values) of cardiac-affected and normally developed embryos after CRISPR-Cas9-mediated knockout of indicated candidate genes versus control (mock injection). Phenotypic proportions of crispants were determined from 54 to 100 embryos and compared to 303 mock-injected control embryos. The mean and standard deviation of the control group were calculated based on seven technical replicates. C Independent replication using base editing: phenotypic distribution (proportion/bars and counts/values) resulting from targeted gene editing mediated by introducing premature termination codons via the cytosine base editor evoBE4max and a set of distinct guide RNAs targeting the same genes as in (B). Phenotypic proportions of editants were determined from 50 to 75 embryos and compared to 105 mock-injected control embryos. The mean and standard deviation of the control group were calculated based on three technical replicates.

Fig. 4. Editing of candidate genes affects embryonic heart rate in medaka embryos.

A Validation workflow including zygotic microinjections using a HdrR (myl7::eGFP; myl7::H2A-mCherry) reporter line, high-throughput image-based heart rate quantification in normally developed injected specimens at 4 days post fertilization (dpf). (B, C) Heart rate distributions and absolute heart rate differences of morphologically normal crispants (n = 13 to 22 embryos) and editants (n = 4 to 22 embryos) compared to mock-injected control embryos (n = 8 to 12 embryos) at 21 °C, 28 °C, and 35 °C with CRISPR-Cas9- (B) and base editor-mediated (C) targeted mutagenesis of candidate genes (heart rate values are listed in Data S2 and sample numbers in Data S3). The significance of heart rate differences between the edited group and its corresponding control was assessed with the two-sided Wilcoxon test; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (p values listed in Data S4). Data is visualized as box plots (median+/− interquartile range between the 25th and 75th percentiles) and overlaid scatter plots of heart rate measurements; knockouts of adprhl1, btbd1, blzf1, phka2, and rrad have temperature-dependent effects on heart rate. Schemes are adapted from a previous publication under a CC BY 4.0 Creative Commons licence.

Fig. 5. Developmental and electrophysiological phenotypes in F0 crispants and F2 mutants.

A Workflow including zygotic microinjections using an HdrR (myl7::eGFP; myl7::H2A-mCherry) reporter line and phenotyping of cardiac-affected crispants 4 days post-fertilization (dpf). B Representative cardiac phenotypes of knockout embryos for all six candidate genes targeted with CRISPR-Cas9 and base editor compared to a control embryo at 4 dpf; bright-field overview of the injected specimen (top; scale bar, 500 µm), close-up image of the heart (bottom; scale bar, 125 µm); cf. movie S2. C Immunostaining of regulatory myosin light chain (myl7) in crispants of btbd1 (n = 5 embryos), blzf1 (n = 6 embryos), and adprhl1 (n = 7 embryos) at 4 days post-fertilization (4dpf) in Cab. In btbd1 and adprhl1 mosaic knockout, the myosin signal is reduced and sarcomeric structure disturbed (filled arrowhead) compared to blzf1, which shows no differences in contrast to control (n = 2 embryos) (unfilled arrowhead); (scale bar, 10 µm). D Heart rhythm analysis in F0 crispants and F2 mutants as depicted by kymographs derived from atrium (A) to ventricle (V) spanning line selection in 10 s time-lapse movies (scale bar, 500 µm). In contrast to the regular rhythm in the control embryo (left), the representative rrad, blzf1, and adprhl1 F0 crispant and F2 mutants (right) display different degrees of atrioventricular block; cf. movie S4. Schemes are adapted from a previous publication under a CC BY 4.0 Creative Commons licence.