2.4. Isolation and culture of neonatal rat cardiomyocytes

Neonatal CMs were isolated by enzymatic disassociation of hearts from 7‐day‐old SD rats according to our published procedure. 14 Briefly, rats were euthanized using overdose of isoflurane (5%) and hearts were extracted. Then, hearts were washed with ice‐cold saline and minced into pieces smaller than 1 mm³ before enzymatic digestion using 1.25 mg/mL trypsin for 3 minutes. The heart pieces were collected further digested using 0.8 mg/mL collagenase II for 30 minutes at 37°C. The digestion supernatant was collected and added with an equal volume of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS before being filtered through a cellular strainer. Then, cell suspension was incubated at 37°C for 90 minutes for differential attachment. After that, cells in the suspension were plated and cultured in DMEM with 10% foetal bovine serum at 37°C in atmosphere with 5% CO₂ for 24 hours. Then, progesterone at various concentrations (10⁻⁹‐10⁻⁴ M) with or without the progesterone blocker RU486 (10⁻⁶ M) was added to the medium to incubate for 24 hours, and then, the cells were fixed with 4% polyformaldehyde for 20 minutes followed by immunostaining. The cells were collected and stored at −80°C for subsequent analysis of protein or mRNA expression. For small interfering RNA (siRNA) experiments, siRNAs against YAP mRNA were transfected into neonatal CMs using Lipofectamine 2000 transfection reagent (Invitrogen) for 24 hours prior to progesterone treatment. Sequences of two different siRNAs are as following: 1# si YAP, 5′‐GCCAUGAACCAGAGGAUCATT ‐3′; 2# si YAP, 5′‐GAUUGAAACAGCAGGAGUUTT‐3′.