Neonatal rat ventricular myocytes (NRVMs) were obtained from the hearts of 1‐2 days old Sprague‐Dawley rats as previously described. 26 Hearts of 1‐ to 3‐day‐old pups were excised and the atria removed. The ventricles were digested 3‐4 times at 37°C for 5 minutes with 0.125% trypsin freshly suspended in calcium‐free HBSS solution, pH 7.4. The supernatants containing isolated cells were collected from each digestion, and an equal volume of serum‐rich medium was added to stop the digestion. Isolated cells were collected and centrifuged at 800 rpm for 5 minutes to separate non‐myocardial cells. The resuspended cells were pre‐plated twice for 30 minutes to further reduce fibroblast contamination. NRVMs were finally plated in 3 cm dishes or 24‐well plates at a density of 0.3‐1 × 106 cells/well, and 15% fetal bovine serum was added into the medium and cultured at 37°C and 5% CO2.

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