Assessing the Metabolic Activity of Whole Regenerating Zebrafish Ventricles Ex Vivo Using an Extracellular Flux Assay

The ability of zebrafish to regenerate their hearts throughout adulthood is partially attributed to metabolic adaptations. Although it has been hypothesized that the fish heart relies significantly on glycolysis, recent studies have uncovered a more complex metabolic profile in which oxidative metabolism arises as an essential component of cardiomyocyte redifferentiation and successful late-stage heart regeneration. In 2025, we adapted a high-throughput method to assess the metabolic profile of whole zebrafish ventricles ex vivo, utilizing the Seahorse assay (extracellular flux assay). This method allows for rapid, real-time assessment of basal and maximal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the XF Mito Stress test on the Seahorse XFe24 analyzer. In this article, we describe a detailed protocol for performing extracellular flux analysis on whole zebrafish ventricles. The ability to quantify the OCR and ECAR in live whole hearts ex vivo will provide the opportunity to elucidate cardiac metabolism at critical timepoints during development, disease progression, and regeneration.