Self-employed regulation of cardiac Kv4.3 potassium channel expression by angiotensin II and JAG2 phenylephrine. promoter in response to ANG II and H2O2. Overexpression of the p50 subunit of NF-B in H9c2 cells reduced scn5a mRNA (77.3%, < 0.01). In conclusion, ANG II can decrease scn5a transcription and current. This effect appears to be through production of H2O2 resulting in NF-B binding to the Na+ channel promoter. published from the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). Quantification of scn5a transcripts by quantitative real-time RT-PCR assay To determine the large quantity of cardiac sodium channel (scn5a) mRNA under the numerous conditions, quantitative real-time RT-PCR was used. The H9c2 cells were treated with H2O2 (20 mol/l), ANG II (100 nmol/l) only, or combined with apocynin (100 mol/l), polyethylene glycol-conjugated catalase (PEG-CAT; 50 U/ml), 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron;10 mmol/l) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-luciferase (phRL-TK; Promega, Madison, CA) was carried out with 0.9 l of Fugene6 chemical transfection reagents (Roche, Indianapolis, IN) following a manufacturers instructions. The serum-free DMEM social press with or without ANG II or H2O2 was changed every 24 h. After becoming cultured for 48 h, the cells were treated with passive lysis buffer (Promega, Madison, Quinupristin CA), and cell components were collected for analysis of firefly and luciferase activities using 100 l of luciferase assay substrate and 100 l of Quit & Glo reagent of the dual-luciferase reporter assay system (Promega). Light emission was quantified inside a Veritas microplate luminometer using Veritas-version 1.4.0 software (Tuener Biosystems, Sunnyvale, CA). Transfection effectiveness of the reporter constructs was controlled by comparison to luciferase activity. The phRL-TK vector minimized any modulation of luciferase manifestation from the experimental conditions since it has been engineered to remove the majority of potential transcription element binding sites. The luciferase activity of the all promoter-constructs was normalized to a pGL3-fundamental promoter-less control transfected simultaneously. Four independent transfection sessions were analyzed, and at each session, transfections were performed in triplicate. Three dual luciferase readings were taken for each transfection experiment. Site-directed mutagenesis of NF-B binding site Disruption of the NF-B binding site was carried out using the QuikChange II XL site-directed mutagenesis kit according to the manufacturers instructions (Stratagene, La Jolla, CA). Briefly, for PCR, 10 ng of pGL3-APS3 was used like a template, and the nucleotide primers outlined were used to mutate Quinupristin the NF-B binding site (the daring as crazy type, the Quinupristin underline as mutant) of pGL3-APS3: NFB-mutCF: 5-GGTGCTGCACTCAGGCCATCCCTATGAGATCCTC-3 and NFB-mutCR: 5-GAGGATCTCATAGGGATGGCCTGAGTGCAGCACC-3. After digestion with value <0.05 was considered statistically significant. RESULTS ANG II and H2O2 dose ranging in H9c2 cells To determine appropriate concentrations of these agents in future experiments, rat H9c2 cardiomyocytes were treated with escalating concentrations of ANG II and H2O2, and the dose-dependent cell viability was identified. H9c2 cardiomyocytes were tolerant of a wide range of ANG II concentrations from 1C500 nmol/l in serum-free medium (Fig. 1< 0.05 when compared with 0 mol/l H2O2. Cardiac Na+ channel current was downregulated by ANG II and H2O2 ANG II and H2O2 reduced Na+ current in a similar manner. Figure 2 demonstrates, compared with control, 100 nmol/l ANG II exposure for 48 h resulted in a 59.6% (5.6%, = 9 for control, = 11 in treated group, < 0.001) decrease in maximum Na+ current. H2O2 exposure (10 mol/l) for 48 h resulted in a similar 53.8% (3.3%, = 9 for control, = 9 in the treated group, < 0.01) decrease in maximum Na+ current. Steady-state channel gating guidelines (> 0.05; Fig. 2, and = 11) and 88.2% (6.6%, = 9) in the ANG II- and H2O2-treated groups, respectively, compared with control (= 9; Fig. 3and and and < 0.05. Open in a separate window.