Each real-time PCR reaction (25 l) contained 2.5 l of cDNA, (for 18 RNA detection, cDNAs were diluted 1000-fold), 12.5 l of 2X SYBR Green Master Mix (Applied Biosystems Inc.) and primers at a final concentration of 350 nmol/l and 900 nmol/l. mediates downstream signaling from NADPH oxidase. Mechanical stretch also downregulates Kv4. 3 3 UTR reporter activity and this requires AT1 receptors and NADPH BI207127 (Deleobuvir) oxidase. Thus, activation of AT1 receptors by Ang II or stretch specifically destabilizes cardiac myocyte Kv4.3 channel mRNA by activating NADPH oxidase. These results link long-term control of cardiac K+ channel gene expression to a physiological reactive oxygen species (ROS) signaling pathway. luciferase (filled bars). For each construct, we normalized the Ang II data to the normalized activities in vehicle-treated myocytes (open bars). Note that the 3 UTR constructs derived from the BI207127 (Deleobuvir) Kv1.5 (PG1.5) and Kv4.2 (PG4.2) were, like PGL3, insensitive to Ang II, while constructs containing most of the Kv4.3 3 UTR were sensitive to Ang II. n=3-9, *** P 0.001. (D) Lack of an Ang II effect on PG4.3B expressed in cardiac fibroblasts (n=3). Similarly, PCR with full length cDNA clones (Clone ID: 4527103 and 30356567, Open Biosystems) was used to introduce 5 XbaI sites and a 3 SalI sites into the 0.8 kb Kv1.5 and the 2 2 kb Kv4.2 3 UTR sequences. These sequences, including their own polyA tracts, BI207127 (Deleobuvir) were digested with restriction enzymes, and then 5 and 3 fragments were digested with XbaI and SalI, respectively. Finally, the complete 3 UTR sequences were reassembled and then cloned into the PGL3 vector between the XbaI and SalI sites to generate the 3 UTR reporter constructs PG1.5 (for the Kv1.5 3 UTR) and PG4.2 (for the Kv4.2 3 UTR). Rat Neonatal Cardiac Myocyte Culture and Treatments Neonatal rat ventricular cardiac myocytes were isolated from collagenase-treated hearts from 1-day old Sprague-Dawley rats as described previously (8). Following dissociation and preplating for 2 hours to remove nonmyocytes, cardiac myocytes were cultured at low density (0.5 x 106 viable cells per well) in 6 well plates containing in Minimal Essential Medium (MEM) supplemented with 5% calf serum (Gibco) and bromodeoxyuridine (100 mol/L) to prevent nonmyocyte proliferation. Twenty PTPSTEP hours BI207127 (Deleobuvir) after plating, the cardiac myocytes were transfected using lipofectamine 2000 at a 1:1 ratio for DNA to lipofection reagent for 4 hours with serum-containing medium. Typically, 3 g of DNA per well was used. However, in experiments comparing various lengths of the Kv4.3 3 UTR (i.e. Figure 1), molar concentration of reporter plasmid was kept constant (1.5-3 g of DNA per well was used). Twenty hours after transfection, the medium was replaced with serum-free MEM supplemented with human insulin (10 g/mL, Sigma), human transferrin (10 g/mL, Sigma), and bovine serum albumin (1 mg/mL, Sigma). For fibroblast cultures, nonmyocytes isolated in the preplating step were retained and cultured as described above except that bromodeoxyuridine was omitted from the medium. Two days later, cultures were subjected to treatment with vehicle, 100 nmol/l Ang II or stretch. For experiments utilizing chemical inhibitors, cells were preincubated with inhibitors or vehicle for 30 minutes before treatment (i.e. Ang II or stretch). For dominant negative subunit experiments, transfection with empty vectors was used as a control. For mechanical stretch experiments, myocytes plated on collagen I-coated six well Flexcell culture plates were subjected to a repetitive cyclical stretch paradigm with a 3 second period and 10% average stretch for 6.5 hours using a Flexcell FX4000 strain unit. Luciferase assay luciferase reporter (pRL-TK, Promega) in a 1:10 ratio was cotransfected with each firefly luciferase reporter construct as an internal, non-inducible reporter standard to account for variation in transfection efficiency and global nonspecific effects. Luciferase activity was measured using Dual-Glo luciferase assay system (Promega). Each experiment included three culture BI207127 (Deleobuvir) wells to generate a single measurement. At least three independent experiments were performed and results are presented with error bars representing the standard error of the mean (SEM). RNA isolation and Quantitative real-time PCR Total myocytes RNA was isolated using TRIZOL Reagent (Invitrogen) after transfection and treatment with vehicle or Ang II. DNA was removed by DNase I treatment prior to cDNA synthesis. Relative quantitation by real-time PCR was carried out using SYBR-green detection of PCR products in real time using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems Inc.)..The ratio between samples without treatment and with treatment is calculated by Relative quantification14. peroxide experiments show that the ASK1 (Apoptosis Signal-regulating Kinase 1)-p38 kinase pathway mediates downstream signaling from NADPH oxidase. Mechanical stretch also downregulates Kv4.3 3 UTR reporter activity and this requires AT1 receptors and NADPH oxidase. Thus, activation of AT1 receptors by Ang II or stretch specifically destabilizes cardiac myocyte Kv4.3 channel mRNA by activating NADPH oxidase. These results link long-term control of cardiac K+ channel gene expression to a physiological reactive oxygen species (ROS) signaling pathway. luciferase (filled bars). For each construct, we normalized the Ang II data to the normalized activities in vehicle-treated myocytes (open bars). Note that the 3 UTR constructs derived from the Kv1.5 (PG1.5) and Kv4.2 (PG4.2) were, like PGL3, insensitive to Ang II, while constructs containing most of the Kv4.3 3 UTR were sensitive to Ang II. n=3-9, *** P 0.001. (D) Lack of an Ang II effect on PG4.3B expressed in cardiac fibroblasts (n=3). Similarly, PCR with full length cDNA clones (Clone ID: 4527103 and 30356567, Open Biosystems) was used to introduce 5 XbaI sites and a 3 SalI sites into the 0.8 kb Kv1.5 and the 2 2 kb Kv4.2 3 UTR sequences. These sequences, including their own polyA tracts, were digested with restriction enzymes, and then 5 and 3 fragments were digested with XbaI and SalI, respectively. Finally, the complete 3 UTR sequences were reassembled and then cloned into the PGL3 vector between the XbaI and SalI sites to generate the 3 UTR reporter constructs PG1.5 (for the Kv1.5 3 UTR) and PG4.2 (for the Kv4.2 3 UTR). Rat Neonatal Cardiac Myocyte Culture and Treatments Neonatal rat ventricular cardiac myocytes were isolated from collagenase-treated hearts from 1-day old Sprague-Dawley rats as described previously (8). Following dissociation and preplating for 2 hours to remove nonmyocytes, cardiac myocytes were cultured at low density (0.5 x 106 viable cells per well) in 6 well plates containing in Minimal Essential Medium (MEM) supplemented with 5% calf serum (Gibco) and bromodeoxyuridine (100 mol/L) to prevent nonmyocyte proliferation. Twenty hours after plating, the cardiac myocytes were transfected using lipofectamine 2000 at a 1:1 ratio for DNA to lipofection reagent for 4 hours with serum-containing medium. Typically, 3 g of DNA per well was used. However, in experiments comparing various lengths of the Kv4.3 3 UTR (i.e. Figure 1), molar concentration of reporter plasmid was kept constant (1.5-3 g of DNA per well was used). Twenty hours after transfection, the medium was replaced with serum-free MEM supplemented with human insulin (10 g/mL, Sigma), human transferrin (10 g/mL, Sigma), and bovine serum albumin (1 mg/mL, Sigma). For fibroblast cultures, nonmyocytes isolated in the preplating step were retained and cultured as described above except that bromodeoxyuridine was omitted from the medium. Two days later, cultures were subjected to treatment with vehicle, 100 nmol/l Ang II or stretch. For experiments utilizing chemical inhibitors, cells were preincubated with inhibitors or vehicle for 30 minutes before treatment (i.e. Ang II or stretch). For dominant negative subunit experiments, transfection with empty vectors was used as a control. For mechanical stretch experiments, myocytes plated on collagen I-coated six well Flexcell culture plates were subjected to a repetitive cyclical stretch paradigm with a 3 second period and 10% average stretch for 6.5 hours using a Flexcell FX4000 strain unit. Luciferase assay luciferase reporter (pRL-TK, Promega) in a 1:10 ratio was cotransfected with each firefly luciferase reporter construct as an internal, non-inducible reporter standard to account for variation in transfection efficiency and global nonspecific effects. Luciferase activity was measured.