High-content screen for cardiovascular modulators in zebrafish (Danio rerio)

Abstract

Over the past two decades, cardiovascular diseases (CVDs) have remained the leading cause of death worldwide. Among these diseases, congenital heart disease is the most common type of human birth defect, affecting 1 in 100 live-born infants. It is crucial to identify new therapeutic targets and treatments for CVDs. Zebrafish are a suitable model for this task, as they can provide new insights into CVDs. Further, their small size and translucency make them an ideal model for high-content drug screenings. In this study, a phenotype-based high-content screening approach in zebrafish was established to identify cardiovascular modulators. Utilizing the ‘Acquifer Imaging Machine‘, 1,280 compounds of the Prestwick Chemical Library were screened for angiogenesis and heartbeat phenotypes. The previously established type III receptor tyrosine kinase inhibitor sunitinib malate, which inhibits angiogenesis in zebrafish, was used as a positive control. Additionally, the competitive 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) inhibitor atorvastatin, which was previously shown to cause severe heart phenotypes in zebrafish, served as a positive control for heart rate (HR) and heart morphology. HR was selected as a screening phenotype under the assumption that morphological and functional changes in the heart during development would result in altered HR. In search of angiogenesis modulators, blood vessel diameters of treated fish were measured for 320 of the 1,280 tested compounds. Application of different filters for known toxicities, previously published results and unknown targets, led to the identification of 11 promising hit compounds. Three of these compounds, namely desonide, oxibendazole, and azlocillin sodium salt, directly targeted angiogenesis by significantly reducing the diameter of intersegmental vessels. The remaining nine compounds affected the diameter of the dorsal aorta and/or dorsal vein. Of the 134 compounds significantly altering HR, 21 compounds repeatedly induced significant HR alterations in two different transgenic zebrafish lines. In this study, two main target clusters were identified for HR modulators: the histaminergic receptor H1 (HRH1) and the glucocorticoid receptor (NR3C1). While HRH1 is commonly associated with allergic reactions, it has also been linked to CVDs. All antagonistic hit compounds targeting HRH1 caused significant bradycardia with moderate to severe dose-dependent teratogenicity. In situ hybridization for various hrh probes revealed that hrh1 was the only detectable hrh in the heart at 3 days post fertilization (dpf), indicating its involvement in heart development. Additionally, the heart analysis tool pyHeart4Fish showed chamber-specific effects of HRH1 antagonists. Collectively, the data suggests that hrh1 plays a role in heart contractility and heart size. However, further investigation on the mechanism of action is necessary to comprehend how Hrh1 is involved in cardiogenesis and what long-term effects HRH1 antagonists can have on the developing embryo to determine drug safety for pregnant women. NR3C1 is a ligand-activated transcription factor known to be involved in inflammation, metabolism, and stress response. In this study involving zebrafish larvae, a group of six NR3C1 agonists was found to significantly increase HR at 48 hours post fertilization. While previous research had identified nr3c1's involvement in heart development in zebrafish, its role in arrhythmias remained unclear. Here, expression of nr3c1 was detected in the larval heart at 5 dpf using in situ hybridization. Further analysis using pyHeart4Fish revealed that NR3C1 agonists induced chamber-specific phenotypes, including atrioventricular block. A homology model of zebrafish Nr3c1 was also developed, which suggested that the agonists known to bind in humans may also bind in zebrafish. These findings support the use of zebrafish as a screening model for cardiovascular phenotypes and highlight the potential role of nr3c1 in CVDs.