Cell culture and treatment
The study was permitted by the Law of the People’s Republic of China on the Protection of Wildlife, and the protocol was approved by the Institutional Animal Care Committee of Shanghai University of medicine & Health Sciences, China (Permit Number: JCLW01-08). Cardiac fibroblasts were isolated from the hearts of 3-day-old neonatal Sprague-Dawley rats and cultured as described previously. Briefly, the hearts were removed and washed with cold PBS. The atria and aorta were discarded. The ventricles were cut open and soaked with 75% ethanol for 30s to inactivate epicardial and endocardial endothelial cells. Then ventricles was finely minced into small pieces and digested with 0.125% trypsin and and 0.5 g/L collagenase II (Invitrogen, Carlsbad, CA) for 6 min in a 37 °C in six consecutive steps. Cells were centrifuged and resuspended in DMEM with 10 % FBS. The cells were seeded at a density of 1×105 cells/ml and incubated for 60 min. the Unattached cells were discarded by washing with PBS, and the attached cells were cultured in DMEM supplemented with 10 % FBS. CFs were identified by their morphology, positive staining for vimentin, and negative staining for α-smooth muscle actin (α-SMA) and von Willebrand factor (vWF). The purity of CFs in this study was more than 97 %. The cells were divided into control, LPS, serelaxin + LPS, serelaxin + LPS+GW9962 and LPS+ GW9962 groups. In LPS group, the cells were induced with LPS (1 μg/ml). In serelaxin and LPS groups, the cells were treated with LPS(1 μg/ml) and serelaxin (100 ng/ml). GW9662 with 100 nM in each groups.