MicroRNAs as Bridging Key Events in Zebrafish Cardiovascular Adverse Outcome Pathways: A Cross-Chemical Evidence...

Zebrafish is among the most widely used vertebrate models for defining how environmental toxicants disrupt cardiovascular development, and the adverse outcome pathway (AOP) framework is the standard tool for organising this mechanistic knowledge for regulatory use. Existing cardiovascular AOPs link molecular initiating events (MIEs), such as aryl hydrocarbon receptor (AhR) activation, to apical outcomes including pericardial edema and early life stage mortality through key events measured largely at the gene and protein level. MicroRNAs (miRNAs) are recurrently dysregulated intermediates in this same causal chain across many toxicant classes, yet remain absent as formally recognised key events in the current cardiovascular AOP network. This review synthesises the zebrafish literature on toxicant-induced cardiovascular malformation, the cardiogenic miRNA circuitry governing chamber formation, looping, valve formation and electrophysiology, and fourteen cardiotoxicity-relevant AOPs catalogued in AOP-Wiki, including both structural and functional cardiac endpoints. Building on this synthesis, we propose a miRNA-bridged key event (miR-BKE) framework that formalises six criteria, four adapted from established weight-of-evidence practice, a fifth grounded in miRNA target-validation standards, and a sixth addressing taxonomic and species variability, for admitting specific miRNAs as quantifiable bridging nodes. We apply this framework, with an explicit numerical scoring rubric, across fourteen toxicant classes spanning industrial, agricultural, pharmaceutical, and consumer-product chemicals, and identify where genomics and small RNA sequencing data are sufficient, partial, predicted only, or entirely absent for formal key event inclusion. A prioritised research agenda ranks these classes by readiness for miR-BKE admission. The review concludes by discussing the framework's taxonomic, chemical-mixture, and methodological limitations, and the priority experiments needed to test it.