Cell isolation and culture

Neonatal CMs from 1-day-old (P1) and 7-day-old (P7) C57BL/6 mice were isolated as previously described.^32,42 Briefly, the ventricles of the mice were isolated, cut into pieces, digested with 0.25% trypsin (Sigma) at 4°C for approximately 12 h, and digested again with type II collagenase (Roche) at 37°C for 15 min two or three times. The supernatant was collected and centrifuged, and the collected cells were resuspended in DMEM/F12 medium (Life Technologies) supplemented with 10% FBS. The cell suspension was subsequently plated onto uncoated 100-mm plastic dishes for 90 min at 37°C and 5% CO₂ in a humidified atmosphere. Due to the differential adhesion between CMs and fibroblasts, the supernatant, composed mostly of CMs, was then collected, resuspended in the aforementioned culture medium, and seeded at the appropriate density.

Adult CMs were isolated from 56- to 70-day-old mice as previously described.^32,42 Briefly, adult hearts were isolated, placed on a Langendorff apparatus, and perfused with calcium-free perfusion buffer for 5 min and digestion buffer for 10 min. The perfusion buffer contained NaCl (113 mM), KCl (4.7 mM), KH₂PO₄ (0.6 mM), Na₂HPO₄ (0.6 mM), MgSO₄ (1.2 mM), Na-HEPES (10 mM), NaHCO₃ (12 mM), KHCO₃ (10 mM), phenol red (0.032 mM), taurine (30 mM), BDM (10 mM), and glucose (5.5 mM, pH 7.0). The digestion buffer contained 15,000 U of type II collagenase (Roche) and 50 μM CaCl₂. Then, the hearts were cut into small pieces and triturated with a Pasteur pipette to separate individual cells. The adult CMs were then centrifuged at a low speed and seeded onto culture slides coated with laminin ( 1 mg/mL Life Technologies, 23017015) in DMEM/F12 medium (Life Technologies) supplemented with 10% FBS. Primary mouse cardiac endothelial cells and fibroblasts were obtained from Cell Biologics (C57-6024 and C57-6049, respectively) and handled according to the company’s instructions. Descriptions of the isolation and quality control analyses of these cells are presented at the company’s website (https://cellbiologics.com/).