Neonatal rat ventricular myocytes (NRVMs) and siRNA knockdowns

Cardiomyocytes were isolated from the left ventricle of 1–2-day-old Sprague-Dawley rats. Lysates were digested with collagenase, and the resulting cell suspension was pre-plated to remove fibroblasts. Myocytes were then plated at a density of 1250 cells/mm in DMEM 4.5 g/L glucose medium containing 10% FBS and 100 µmol/L bromodeoxyuridine. For siRNA knockdown experiments, NRVMs were transfected 36 hours after plating with siRNA constructs (Sigma) using Lipofectamine RNAiMax (Invitrogen) in Optimem (6h). Experiments were launched 24h after knockdown. For hypertrophy studies, cells (48h after plating) were exposed to serum-free DMEM containing: phenylephrine (PE) [50 µM], endothelin-1 (ET-1) [100 nM], insulin growth factor-1 (IGF1) [10 nM], or 50% hypo-osmotic solution. HDAC inhibitors were employed as follows: apicidin (Api) at 0.2 µM and 1 µM; trichostatin A at 60 nM.(8)