2.2. Cardiac fibroblast isolation

4 or 12 weeks after sham or ACF surgery, adult rat LV fibroblasts were isolated by recirculating perfusion buffer supplemented with 1 mg/ml collagenase type II (Invitrogen, CA) as previously described [1]. Briefly, the heart was perfused with a buffer (120 mM NaCl, 15 mM KCl, 0.5 mM KH₂PO₄, 5 mM NaHCO₃, 10 mM HEPES, and 5 mM glucose, at pH 7.0) for 5 min and digested with perfusion buffer containing 1 mg/ml collagenase II for 30 min at 37 °C. The right ventricle and atria were removed before the perfused-heart was minced. The cell suspension was then mixed with stop buffer (perfusion buffer containing 10 mg/ml bovine serum albumin) to prevent further digestion. The cell suspension was added to a mesh cell collector and the flow-through was centrifuged at 80 g for 3 min to remove most of the cardiomyocytes. The supernatant containing mainly cardiac fibroblasts was centrifuged at 400 g for 8 min then resuspended in DMEM supplemented with antibiotics (penicillin/streptomycin, 1%), L-glutamine ascorbate and 10% FBS. Cells were subjected to differential plating on uncoated cell culture dishes (10 cm diameter) for 90 min. Non-adherent cells (mostly cardiomyocytes, endothelial and smooth muscle cells) were removed. We have demonstrated >95% purity of the prep with very little myofibroblast differentiation [1]. Cultured cells by this protocol routinely showed positive staining for anti-vimentin (Millipore #AB5733; 1:500) and no immunostaining with antibodies to anti-smooth muscle alpha-actin or fibronectin (1:100 dilutions, Sigma-Aldrich, MO). Adult rat cardiac fibroblasts were used at passages 1 and 2 in the current study.