Investigating Phenylephrine-Induced Cardiac Hypertrophy

The experiments were performed as described previously [16 (link),32 (link)]. H9C2 were treated with 10⁻⁷ M phenylephrine (PE, Sigma-Aldrich Corporation, St. Louis, MO, USA) for 24 h. In some experiments, cells were treated with the synthetic peptide TAT-RH, which only reproduce the RH domain of GRK5, as described previously [18 (link)] or transfected with plasmids encoding for GRK5-NT or its mutant in the amino-terminal calmodulin binding. At the end of the treatment, cells were lysed in RIPA/SDS buffer, and protein concentration was determined by using Pierce BCA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) [33 (link),34 (link)]. Total extracts were electrophoresed by SDS/PAGE and transferred to nitrocellulose [35 (link)]. The antibodies anti-NFATc4 (B-2) (SC-271597), GATA-4 (G-4) (sc-25310), p-NFATc4 (80.S168/170) (sc-135770), p-GATA-4 (H-4) (sc-377543), β-actin (C-4) (sc-47778), and histone H3 (FL-136) (sc-10809) were from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc, Dallas, TX, USA). In some experiments, nuclear proteins were isolated from heart samples as previously described [16 (link)]. Densitometric analysis was performed using Image Quant 5.2 software (Molecular Dynamics Inc., Caesarea, Israel). Images are representative of at least three independent experiments quantified and corrected for appropriate loading control.