Using Zebrafish for Investigating the Molecular Mechanisms of Drug-Induced Cardiotoxicity

Over the last decade, the zebrafish (Danio rerio) has emerged as amodel organismfor cardiovascular research.Zebrafish have several advantages over mammalian models. For instance, the experimental cost of using zebrafish is comparatively low; the embryos are transparent, develop externally, and have high fecundity making them suitable for large-scale genetic screening. More recently, zebrafish embryos have been used for the screening of a variety of toxic agents, particularly for cardiotoxicity testing. Zebrafish has been shown to exhibit physiological responses that are similar to mammals after exposure to medicinal drugs including xenobiotics, hormones, cancer drugs, and also environmental pollutants, including pesticides and heavy metals. In this review, we provided a summary for recent studies that have used zebrafish to investigate themolecularmechanisms of drug-induced cardiotoxicity. More specifically, we focused on the techniques that were exploited by us and others for cardiovascular toxicity assessment and described several microscopic imaging and analysis protocols that are being used for the estimation of a variety of cardiac hemodynamic parameters.Huseyin C. Yalcin is supported by Qatar National Research Fund (QNRF), National Priority Research Program NPRP 10-0123-170222,and Qatar University internal funds,QUUGBRC-2017-3 and QUST-BRC-SPR\2017-1. The publication of this article was partially funded by the Qatar National Library

AEO-7 surfactant is “super toxic” and induces severe cardiac, liver and locomotion damage in zebrafish embryos

© 2020, The Author(s). Background: Fatty alcohol polyoxyethylene ether-7 (AEO-7), a non-ionic surfactant, has recently been receiving extensive attention from the ocean pipeline industry for its ability to inhibit corrosion. However, the present lack of information concerning the potential environmental toxicity of AEO-7, especially towards aquatic organisms, is a major impediment to its wider application. Here, we assess potential adverse effects of AEO-7 on zebrafish embryos employing a variety of assays, including (i) a mortality/survival assay which allowed the median lethal concentration (LC50) to be calculated; (ii) a teratogenicity assay on the basis of which the no observed effect concentration (NOEC) was determined; and (iii) specific assays of cardiotoxicity, neurotoxicity (based on locomotion), hematopoietic toxicity (the level of hemoglobin as revealed by o-dianisidine staining) and hepatotoxicity (liver steatosis and yolk retention examined by staining with Oil Red O). Results: AEO-7 caused mortality with a calculated LC50 of 15.35 μg/L, which, according to the U.S. Fish and Wildlife Service (USFWS) Acute Toxicity Rating scale, should be considered “super toxic”. Although at its NOEC (0.8 μg/L), there were no signs of significant teratogenicity, cardiotoxicity, or hemopoiesis toxicity, 3.2 µg/L AEO-7 exerted dramatic detrimental effects on organ development. Conclusion: On the basis of these findings, we recommend that the industrial usage and environmental impact of AEO-7 be re-evaluated and strictly monitored by environmental and public health organizations

PGAP3 Associated with Hyperphosphatasia with Mental Retardation Plays a Novel Role in Brain Morphogenesis and Neuronal Wiring at Early Development

Recessive mutations in Post-GPI attachment to proteins 3 (PGAP3) cause the rare neurological disorder hyperphosphatasia with mental retardation syndrome 4 type (HPMRS4). Here, we report a novel homozygous nonsense mutation in PGAP3 (c.265C>T-p.Gln89*), in a 3-year-old boy with unique novel clinical features. These include decreased intrauterine fetal movements, dysgenesis of the corpus callosum, olfactory bulb agenesis, dysmorphic features, cleft palate, left ear constriction, global developmental delay, and hypotonia. The zebrafish functional modeling of PGAP3 loss resulted in HPMRS4-like features, including structural brain abnormalities, dysmorphic cranial and facial features, hypotonia, and seizure-like behavior. Remarkably, morphants displayed defective neural tube formation during the early stages of nervous system development, affecting brain morphogenesis. The significant aberrant midbrain and hindbrain formation demonstrated by separation of the left and right tectal ventricles, defects in the cerebellar corpus, and caudal hindbrain formation disrupted oligodendrocytes expression leading to shorter motor neurons axons. Assessment of zebrafish neuromuscular responses revealed epileptic-like movements at early development, followed by seizure-like behavior, loss of touch response, and hypotonia, mimicking the clinical phenotype human patients. Altogether, we report a novel pathogenic PGAP3 variant associated with unique phenotypic hallmarks, which may be related to the gene’s novel role in brain morphogenesis and neuronal wiring

The Arrhythmogenic E105A CAM Mutation Dysregulates Normal Cardiac Function in Zebrafish by Altering CAM-Ca2+ and CAM-RyR2 Interactions

Calmodulin (CaM) is a multifunctional calcium (Ca2+)-binding messenger that directly interacts with the cardiac ryanodine receptor 2 (RyR2), a large transmembrane Ca2+ channel that mediates Ca2+ release from the sarcoplasmic reticulum to activate cardiac muscle contraction. Genetic studies have reported CaM missense mutations in individuals with history of life-threatening arrhythmogenic heart disorders. A recent clinical report identified a novel, long QT syndrome (LQTS)-associated CaM mutation (E105A), in a child, who experienced an aborted first-episode of cardiac arrest. Herein, to determine the functional consequences of the E105A mutation in vivo, we introduced this mutation into human CaM sequence and we injected synthetic mRNA encoding CaMWT and CaME105A into zebrafish embryos. Although expression of CaMWT and CaME105A proteins in zebrafish did not affect the normal embryo development, a slight change in the heart morphology was observed, with ∼31.5% of the CaME105A-injected zebrafish exhibiting extended hearts. Furthermore, analysis of the cardiac activity of the zebrafish ventricle revealed that CaME105A mutant-injected larvae displayed irregular pattern of heart beating in comparison to the median of the CaMWT and control groups, resulting to an increased arrhythmic potential in these embryos. In addition, the average heart rate was significantly increased in this group (∼160.5 beats per minute (bpm) vs ∼152.5 bpm of control group). In vitro Ca2+-binding studies revealed that the C-domain of CaME105A mutant exhibited a ∼10-fold reduced Ca2+-binding affinity compared to CaMWT. Finally, the functional effect of E105A mutation on RyR2 activity was assessed by a [3H]ryanodine binding assay and suggested that CaME105A mutant shows a dramatically reduced inhibition of ryanodine binding to RyR2 compared to CaMWT. Our findings suggest that LQTS-associated E105A CaM mutation dysregulates normal cardiac function in zebrafish by altering both CaM-Ca2+ and CaM-RyR2 interactions

The link between glycemic control measures and eye microvascular complications in a clinical cohort of type 2 diabetes with microRNA-223-3p signature

Background: Type 2 diabetes (T2D) is a critical healthcare challenge and priority in Qatar which is listed amongst the top 10 countries in the world, with its prevalence presently at 17% double the global average. MicroRNAs (miRNAs) are implicated in the pathogenesis of (T2D) and long-term microvascular complications including diabetic retinopathy (DR). Methods: In this study, a T2D cohort that accurately matches the characteristics of the general population was employed to find microRNA (miRNA) signatures that are correlated with glycemic and β cell function measurements. Targeted miRNA profiling was performed in (471) T2D individuals with or without DR and (491) (non-diabetic) healthy controls from the Qatar Biobank. Discovery analysis identified 20 differentially expressed miRNAs in T2D compared to controls, of which miR-223-3p was significantly upregulated (fold change:5.16, p = 3.6e−02) and positively correlated with glucose and hemoglobin A1c (HbA1c) levels (p-value = 9.88e−04 and 1.64e−05, respectively), but did not show any significant associations with insulin or C-peptide. Accordingly, we performed functional validation using a miR-223-3p mimic (overexpression) under control and hyperglycemia-induced conditions in a zebrafish model. Results: Over-expression of miR-223-3p alone was associated with significantly higher glucose (42.7 mg/dL, n = 75 vs 38.7 mg/dL, n = 75, p = 0.02) and degenerated retinal vasculature, and altered retinal morphology involving changes in the ganglion cell layer and inner and outer nuclear layers. Assessment of retinal angiogenesis revealed significant upregulation in the expression of vascular endothelial growth factor and its receptors, including kinase insert domain receptor. Further, the pancreatic markers, pancreatic and duodenal homeobox 1, and the insulin gene expressions were upregulated in the miR-223-3p group. Conclusion: Our zebrafish model validates a novel correlation between miR-223-3p and DR development. Targeting miR-223-3p in T2D patients may serve as a promising therapeutic strategy to control DR in at-risk individuals

Biallelic NAA60 variants with impaired N-terminal acetylation capacity cause autosomal recessive primary familial brain calcifications

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning