Metformin Attenuates Cardiac Developmental Toxicity in High-Glucose-Exposed Zebrafish Larvae: Oxidative Stress, Hif-1α-Associated Metabolic Gene Alterations, and Wnt Signaling Changes

Background: Maternal hyperglycemia is associated with embryonic developmental abnormalities and congenital heart defects, yet the mechanisms and effective interventions remain unclear. We examined high-glucose-induced cardiac developmental injury in zebrafish and whether metformin co-administered with high glucose is associated with protection. Methods: Wild-type AB and Tg(myl7:EGFP) zebrafish (Danio rerio) embryos were exposed to control medium, high glucose (150 mM), or high glucose plus metformin (0.75, 3.0, or 6.0 mM); metformin alone was not tested. Hatching and survival rates, cardiac morphology and function, larval locomotor activity, oxidative-stress markers, and cardiac developmental and pathway transcripts/proteins were assessed. Results: Under high-glucose exposure conditions (150 mM), hatching and survival were reduced, cardiac morphology/function and locomotor activity were altered, hand2, gata4, tbx1, and tbx5 were downregulated, Gata4 was decreased, oxidative stress was increased, and Hif-1α expression and downstream metabolic targets were increased. Wnt-related readouts were heterogeneous: Gsk3β Ser9 phosphorylation, β-catenin and Daam1 decreased, whereas Dvl2 increased. Metformin was associated with improved survival, cardiac phenotypes, and locomotor outcomes, together with partial molecular restoration. As an osmolarity-matched control was not included, these phenotypes cannot be attributed exclusively to glucose-specific effects. Conclusions: High-glucose exposure conditions are associated with cardiac developmental injury and pathway dysregulation, and metformin shows protective associations. These findings are not causal, do not distinguish glucose-specific effects from hyperosmotic stress, and require osmolarity-matched controls, functional metabolic assays, and Hif-1α/Wnt perturbation studies.