Zebrafish Cardiac Regeneration Model

The following zebrafish strains were used in this study: wild-type AB (Oregon) strain, Ekkwil (EK) strain, transgenic strain cmlc2::DsRed2-nuc [26 (link)] to visualize cardiomyocytes nuclei, and transgenic strain cmlc2::EGFP [27 (link)] to analyze the injured area on the whole hearts. Fish aged 6-18 months were anesthetized in 0.1% tricaine (Sigma Aldrich) and placed ventral side up in a damp sponge. A small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. The tip of the cryoprobe was 6 mm long with a diameter of 0.8 mm, the handle of the cryoprobe was 4 cm long with a diameter of 8 mm and was covered with a plastic surface. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. For heart resection surgeries, ventricular muscle was removed at the apex with iridectomy scissors as previously described [15 (link)]. Animals were allowed to regenerate for various times at 26.5°C. Experimental research on animals has been approved by the cantonal veterinary office of Fribourg.