2.12 Cardiomyocyte isolation and calcium imaging

Cardiomyocytes were isolated using a Langendorff perfusion system, as described previously (Sun et al., 2021). After perfusion, only RA was removed, and cardiomyocytes from the RA were used for calcium imaging. Calcium imaging was performed according to a previous publication (Sun et al., 2021). In brief, before contractility and calcium analyses, calcium was re-introduced into isolated cardiomyocytes by treating the cells with a series of 10 mL of 2,3-butanedione monoxime-free perfusion buffers containing 100 nmol/L, 400 nmol/L, 900 nmol/L, and 1.2 μmol/L CaCl₂. At each step, cardiomyocytes were settled down by gravity for 10 min at room temperature before cells were transferred to the next buffer with a higher calcium concentration. Cardiomyocytes were loaded with 5 μmol/L Rhod-4™, AM (21121, AAT Bioquest, United States) for 20 min. Then, the cells were washed with normal Tyrode’s solution (NaCl, 140 mmol/L; KCl, 4 mmol/L; MgCl₂, 1 mmol/L; CaCl₂, 1.8 mmol/L; glucose, 10 mmol/L; and HEPES, 5 mmol/L, pH = 7.4, adjusted with NaOH) for 20 min. The cells were subsequently settled in a laminin-coated glass-bottomed flow chamber at 30°C for 10 min and electrically stimulated at 1 Hz to produce steady-state conditions. Finally, calcium signals were acquired through confocal line scanning using a ×63 objective. A line scan was positioned along the long axis of the cell in the cytosol, avoiding the nuclear area. Calcium signals were quantified manually using Fiji 2.9.0.