Culturing and Treating Cardiac Cell Lines

Mouse atrial cardiomyocyte HL-1 cells were a gift from Dr. William Claycomb at Louisiana State University Medical Center. HL-1 cells were cultured at 37°C and 5% CO₂ on surfaces pre-treated with 12.5 μg/ml bovine fibronectin in 0.02% gelatin solution and grown in Claycomb medium supplemented with Fetal Bovine Serum (10%) (both from Sigma-Aldrich, St. Louis, MO), Penicillin/Streptomycin (100U/ml: 100mg/ml), Norepinephrine (100U/ml) and L-Glutamine (2mM) (GIBCO-Invitrogen) as described previously [38 (link), 41 (link)]. hCAVSMCs were purchased from GIBCO-Invitrogen Cell culture (Carlsbad, CA) and were cultured according to manufacturer’s instructions at 37°C and 5% CO₂ in Medium 231 supplemented with Smooth Muscle Growth Supplement (SMGS, Life Technologies, Cat. No. S-007-25). β-blockers Aten, Met and Car and Ang II were purchased from Sigma-Aldrich, and Neb was a gift from Forest Laboratories Inc. (New York). Losartan (AT1R inhibitor), PD123319 (AT2R inhibitor), CGP42112a (partial AT2R agonist), SR59230A (β-3 AR specific blocker), U73122 (Phospholipase C inhibitor) and isoproterenol (β-AR activator) were purchased from Tocris Bioscience (Bristol, UK); NP-6A4 was a gift from Novopyxis Inc. (Boston).