Cryoinjury procedure

Cryoinjury was performed as previously described^60,61. Briefly, fish were presedated in water containing 0.03 mg ml⁻¹ Tricaine (PHARMAQ, pH 7). The concentration was then increased to 0.168 mg ml⁻¹ for anesthesia. Fish were placed with the ventral side facing up in a foam holder under a dissecting scope. To access the heart, a small incision was made through the body wall and the pericardium, using microdissection forceps and scissors. Once the pericardial sac was opened, the heart ventricle was exposed by gently compressing the abdomen. Excess water was carefully removed by blotting with tissue paper, not allowing the fish skin to dry. Then, a stainless steel cryoprobe precooled in liquid nitrogen was applied to the ventricular wall for 20 s. Fish were then placed in a tank of fresh system water with 1.5 mg l⁻¹ morphine sulfate for 6 h⁶²; for the analysis of scRNA-seq data presented here, we also included two morphine-treated and two control hearts from Lelek et al.⁶² (~20,000 cells, about 10% of the total data set). Reanimation was enhanced by gill oxygenation, in which water around the gills was aerated by pipetting for a couple of minutes. To investigate the effects of the cryoinjury depth, fish were injured using the same procedure, but the cryoprobe was applied for 5 s, 10 s, 15 s, 20 s and 25 s, respectively. Established protocols use 20 s of cryoprobe application^60,61. For all injuries, a timer was used to assure the reproducible timing of the cryoprobe contact with the heart tissue. Hearts were analysed at 3, 7, 15 and 30 d.p.i.