Esketamine induces embryonic and cardiac malformation through regulating the nkx2.5 and gata4 in zebrafish

Esketamine (EK) has been widely used in the treatment of depression, but the effects of EK prenatal treatment on embryonic heart development have been rarely reported. This study assesses the effects of varying concentrations of EK on embryonic development and cardiogenesis to determine the teratogenic concentration in the zebrafish model, centering on the interaction with the genes nkx2.5 and gata4 to elucidate the mechanism cardiac morphogenesis. Zebrafish embryos were classified into six distinct groups and exposed to either a vehicle or EK to ascertain the half lethal concentration (LC50) at 48 hours post-fertilization (hpf) and 72hpf through enumerating statistics on mortality rates. Embryonic and heart morphologies were assessed utilizing live embryo imaging techniques and stereo microscopy. Nkx2.5 and gata4 were identified via whole-mount in situ hybridization. Exposure to EK results in concentration- and time- dependent significant teratogenic effects on zebrafish embryos. The 48h- and 72h-LC50 of EK for zebrafish embryos were 0.31 (95% CI, 0.22, 0.38) mg·mL-1 and 0.17 (95% CI，0.11, 0.24) mg·mL-1, respectively. A significant reduction in heart rates and body length were observed and the distance between the sinus venosus and bulbar artery (SV-BA) was also found expanded, the pericardial edema area showed significant swelling, and the body axis curvature was more pronounced in the EK exposure groups. WISH analysis showed nkx2.5 staining intensity significantly decreased, while gata4 expression notably increased in direct proportion to EK concentration increase. Our findings suggest that exposure of zebrafish embryos to EK leads to embryonic and cardiac malformations, primarily due to the down-regulation of nkx2.5 and the over-expression of gata4. The insights advocate to maintain equilibrium and a compensatory mechanism in the spatiotemporal regulation of gene expression is of paramount importance.