Cell isolation and culture

WKY and SHR (n = 5) were anaesthetized by 2% isoflurane and euthanized by cervical dislocation. Primary CF were isolated from rat whole hearts by the following protocol. Hearts were extracted with sterile pliers and placed in tubes with sterile DMEM High Glucose, with 1% penicillin/streptomycin, 0.5% gentamicin, and 1% amphotericin B (EuroClone). Cardiac tissue was transferred onto a tissue culture glass dish and minced using disposable sterile scalpels. Tissue fragments were incubated at 37 °C for 15 min in 10 ml of digestion buffer, composed by phosphate buffer saline (PBS, Euroclone), 1% penicillin/streptomycin, 1% Amphotericin B, and Liberase TH Research Grade Blendzyme (Roche). After washing 3 times with PBS, digested tissue fragments were cultured on vitronectin-coated dishes at 37 °C, 5% CO₂ in DMEM High Glucose (EuroClone) supplemented with 15% Fetal Bovine Serum (FBS, EuroClone), 2 mM l-glutamine, 200 U/ml penicillin, 200 μg/ml streptomycin, and 0.5% Amphotericin B. For the signaling experiments, CF were serum starved and treated with 5 ng/ml TGF-β1 (Peprotech) for 30 min, while, for the other experiments, cell were treated with 5 ng/ml TGF-β1 and 0.5 μM cilengitide (MedChem Express) for 48 h. For stiffness-dependent experiments, two substrates of known stiffness (4.1 kPa, 30 kPa) were produced by varying the content of acrylamide and the ratio acrylamide/bisacrylamide, according to a previously published method [26]. Briefly, small drops (70 μl) of polyacrylamide solution were deposited onto glass slides (30 mm diameter); glass coverslips, previously treated with Surfacil (Pierce) were placed on the top of the solution drops and kept under nitrogen flow, until thin uniform polyacrylamide gels were formed. Finally, polyacrylamide gels surface was chemically activated (Sulfo-SANPAH-Pierce), and coated by vitronectin solution (ThermoFisher, #A14700) incubation O/N at 4 °C. Substrate sterilization was performed by 30 min UV (254 nm) light exposure.