Neonatal Rat Cardiac Myocyte Hypoxia Assay

Neonatal rat cardiac myocytes (NRCMs) were prepared from the ventricles of 2-day-old SD rats^{51 (link)}. All experiments using NRCMs were approved by the ethics committees at the National Institutes of Natural Sciences (Japan) and performed according to approved protocol code: 23A015. NRCMs were seeded on a matrigel-coated glass bottom dish and cultured in DMEM (low glucose) supplemented with 2% FBS. Two days after plating, NRCMs were incubated under normoxia or hypoxia (1% O₂) for 6 h. TT or TTS (10 µM) was added during the last 30 min of normoxia or hypoxia. Untreated cells served as controls.
For H₂S₂ imaging, NRCMs were incubated with 5 µM SSP4 in HBSS containing 0.04% Pluronic F-127 and 2 μg/mL Hoechst for 30 min^{37 (link)}. NRCMs were washed with HBSS three times and imaged by the BZ-X710 (Keyence). The SSP4 fluorescence intensities from the cell images were calculated using Fiji and ImageJ (National Institutes of Health). Data are expressed as mean ± standard error of the mean (SEM). Statistical evaluations were performed on GraphPad Prism with ordinary one-way ANOVA.
For H₂S imaging, cells (without TT/TTS treatment) were incubated with 2.5 µM SF7-AM in HBSS containing 2 μg/mL Hoechst for 30 min. NRCMs were washed with HBSS three times and imaged by the Keyence BZ-X710. The results are shown in the Supplementary Fig. 8.