Exp 3174

Cultured cardiomyocytes from 1‐ or 2‐day‐old rats were isolated, subjected to Percoll^® gradient centrifugation, and plated on deformable membranes coated with collagen‐IV on Bioflex plates (Flexcell International Corp, Hillsborough, NC, USA) in MEM medium supplemented with 5% calf serum and maintained at 37 °C in humidified air with 5% CO₂. Culture media was changed to serum‐free media 24 h prior to initiation of the experiments. Cardiac fibroblasts were obtained as described previously and cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS 23. Cells were passaged 1 : 4 at 80% confluence by trypsinization and minimal pipetting. Thirty minutes prior to stretch, LOS (200 nmol·L⁻¹), SIM (10 μmol·L⁻¹), or Exp‐3174 (MERCK & Co., Inc.) (200 nmol·L⁻¹) was added to the culture medium. The cultured cardiomyocytes were subjected to 20% equiaxial sustained cyclic stretch (1 Hz) with an FX‐2000™ (Flexcell International Co.) strain unit equipped with loading posts 24.

After carrying out dose-response curves to human recombinant TGF-β₁ (RαD systems), adult human dermal fibroblasts (HDFa line; GIBCO) were incubated for 24 hours with or without 10⁻⁴ μg/mL, in the absence or presence of the metabolites EXP3174 and EXP3179 (provided by Merck & Co, Inc.) at a range of concentrations. In addition, HDFa cells were co-incubated with EXP3179 in the absence or presence of the following compounds: the inhibitors of PPAR-γ (G3335) and phosphatidylinositol 3-kinase (PI3K) pathway (LY294002) and the activators of protein kinase-C (PKC) (Phorbol 12-myristate 13-acetate [PMA]) and cyclo-oxygenase-2-pathway (lipopolysaccharide [LPS]).