Isolation and culture of rat primary neonatal cardiomyocytes

Neonatal rat cardiomyocytes were isolated from two‐ to three‐day‐old of Sprague Dawley rat neonates using the protocol described previously 17, 18. All experiments involving animals were performed in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and were approved by the University of Manchester Ethics Committee. For NRCMs isolation, extracted neonatal hearts were preserved into ice‐cold filter‐sterilized ADS solution (116 mm NaCl, 20 mm HEPES, 1 mm NaH₂PO₄, 5.5 mm glucose, 5.5 mm KCl, 1 mm MgSO₄, pH 7.35 adjusted with NaOH). The top section of the heart was excised to remove the atria. Then, the remaining part of the hearts were cut into half and were incubated in ADS solution containing 0.6 mg·mL⁻¹ collagenase A (Roche Applied Bioscience, Mannheim, Germany) and 0.6 mg·mL⁻¹ pancreatin (Sigma‐Aldrich, St. Louis, MO, USA) at 37 °C for 7 min in a shaking water bath. After seven successive cycles of digestion, cells pooled from all digestions were centrifuged at 335 g for 5 min. Cells were plated onto 100‐mm Falcon tissue culture dishes for 90 min in preplating medium (68% DMEM, 17% M199 medium, 10% horse serum, 5% FBS and 2.5 μg·mL⁻¹ amphotericin B) to attach cardiac fibroblasts. Most of the cardiomyocytes remained floating and were collected afterwards. Cardiomyocytes were then plated on BD Falcon Primaria plates or on laminin‐coated cover slips in plating medium containing 68% DMEM, 17% M199 medium, 10% horse serum, 5% FBS, 2.5 μg·mL⁻¹ amphotericin B and 1 μm BrdU (5‐bromo‐2‐deoxyuridine). The following day, NRCMs were washed twice using PBS and then cultured in maintenance media (80% DMEM, 20% M199, 1% FBS, 2.5 μg·mL⁻¹ amphotericin B and 1 μm BrdU) at 37 °C.