Cardiac Fibroblast Isolation and Phenotype Modulation

Fbs from the LV were isolated by a chunk enzymatic digestion protocol as described before^{20 (link)}. After first passage, Fbs were seeded at a density of 3000 cells/cm² and cultured in DMEM (ThermoFisher) medium with 10% foetal bovine serum (FBS) and 1% of penicillin/streptomycin (Invitrogen). To induce specific Fb phenotypes, cells were either treated for 6 days with recombinant human TGF-β1 (400 pmol/L; PeproTech) or with SD-208 (3 μmol/L; Sigma-Aldrich), a specific TGF-β-receptor-I (TGF-β-RI) kinase inhibitor to respectively promote and suppress differentiation. After 6 days, the phenotype was assessed by protein expression and morphology, and functionally as the capacity for proliferation, migration, and contraction of a floating gel. These pre-treated Fbs and MyoFbs were then processed for co-culture with freshly isolated CMs. The supplemental material and Figures S1 detail the preparation and properties of the fibroblastic cells and the subsequent setup of the co-cultures.