Ventricles were dissected from neonatal (1–2 d) Sprague-Dawley rat hearts and dissociated by serial digestion with 0.4 mg/ml collagenase and 0.6 mg/ml pancreatin sterile digestion buffer (116 mM NaCl, 20 mM HEPES, 0.8 mM Na
2HPO
4, 5.6 mM glucose, 5.4 mM KCl and 0.8 mM MgSO
4, pH 7.35). The first digestion supernatant (5 min, 37°C, 160 cycles/min in a shaking waterbath) was removed and discarded. Cell suspensions from subsequent digestions (20 min, 2×25 min, 20 min, 10 min; 37°C 136 cycles/min shaking) were recovered by centrifugation (5 min, 60×g) and the cell pellet resuspended in plating medium (Dulbecco's modified Eagle's medium (DMEM)/medium 199 [4:1 (v/v)], 15% (v/v) FCS, 100 units/ml penicillin and streptomycin). The cells were pre-plated on plastic tissue culture dishes (30 min) to remove non-cardiomyocytes. For biochemistry and molecular biology experiments, non-adherent viable cardiomyocytes were plated at a density of 4×10
6 cells/dish on 60 mm Primaria dishes pre-coated with sterile 1% (w/v) gelatin (Sigma-Aldrich UK). After 18 h myocytes were confluent and beating spontaneously. For immunostaining experiments, cardiomyocytes were plated at 1.5×10
6 cells/dish on 35 mm Primaria dishes containing glass coverslips pre-coated with sterile 1% (w/v) gelatin followed by laminin (20 µg/ml in PBS; Sigma-Aldrich UK). The plating medium was withdrawn and cells were incubated in serum-free maintenance medium (DMEM/medium [4:1 (v/v)], 100 units/ml penicillin and streptomycin) for a further 24 h.
Cardiomyocytes were unstimulated (Controls), exposed to PD184352 (2 µM), a cell-permeable form of the exoenzyme C3 transferase from
Clostridium botulinum (C3T; 1 or 5 µg/ml) or ET-1 (100 nM), or exposed to ET-1 following pretreatment with PD184352 (10 min) or C3T (120 min). PD184352 (Alexis Biochemicals, Enzo Life Sciences) was prepared as a stock solution in DMSO (4 mM). Cell-permeable C3T (Cytoskeleton Inc., Cat. no. CT03) was resuspended in 66% (v/v) glycerol (0.02 mg/ml). This represents highly purified C3T linked to a cell penetrating moiety though a disulfide bond that allows rapid uptake into cells. In the cytoplasm, the disulfide bond is reduced and the C3T diffuses through the cell. ET-1 (Bachem UK) was prepared as a stock solution in water (0.1 mM). ET-1, PD184352 or C3T were added directly to the tissue culture medium. Unless otherwise stated, incubation times with ET-1 were dependent upon the time at which individual signaling components are substantially activated: 30 s for RhoA.GTP loading [25] (
link), 5 min for activation of ERK1/2 [26] (
link) or p38-MAPKs [27] (
link), 15 min for activation of JNKs [28] (
link) and 1 h for expression of immediate early genes [10] (
link). For inhibitor studies, cells were pre-treated with inhibitor for a minimum period required to provide adequate inhibition of the pathways (10 min for PD184352; 2 h for C3T) prior to addition of ET-1 for the appropriate times. Cells were also exposed to inhibitor alone for the full duration of the incubation time. All samples were harvested together at the end of the experiment.
Marshall A.K., Barrett O.P., Cullingford T.E., Shanmugasundram A., Sugden P.H, & Clerk A. (2010). ERK1/2 Signaling Dominates Over RhoA Signaling in Regulating Early Changes in RNA Expression Induced by Endothelin-1 in Neonatal Rat Cardiomyocytes. PLoS ONE, 5(4), e10027.