Gsk1016790a

TRPV4, ROCK1, ROCK2, and paxillin (PXN) antibodies were purchased from Abcam (Cambridge, MA, USA). RhoA, RhoB, RhoC, CDC42, RAC1/2, LIMK, phospho-LIMK, cofilin, phospho-cofilin, MLC, phospho-MLC, MYPT, and phospho-MYPT antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). GAPDH were purchased from Proteintech (Rosemont, IL, USA). Rho pathway antagonist Y27632, calcium chelator BAPTA-AM, TRPV4 agonist GSK1016790A, and its antagonist HC067047 were purchased from Selleck (Shanghai, China). Opti-MEM medium and Lipofectamine RNAiMAX reagent, Fluo-4 AM, Rhodamine Phalloidin, puromycin, and DAPI were purchased from Invitrogen (Massachusetts, USA).

Atrial fibroblasts were isolated from the atrial tissues of sham and SP rats, as described previously (6 (link)). Briefly, tissues were digested with 100 U/mL collagenase (Type II) and 0.1% trypsin (Amresco) for 8 consecutive 7- to 10-minute treatment periods at 37°C. Atrial fibroblasts were pelleted at 1000 rpm for 10 minutes and resuspended in DMEM (Thermo Fisher Scientific) supplemented with 10% FBS (Thermo Fisher Scientific), 100 U/mL penicillin (Servicebio), and 100 μg/mL streptomycin (Servicebio). Experiments were performed with cells from the first and second passages. Before cells were treated with different chemical interventions, the FBS in the medium was deprived for 24 hours to induce growth arrest. The cells were then treated with TRPV4 antagonist GSK2193874 (300 nM) or TRPV4 agonist GSK1016790A (300 nM, Sigma-Aldrich) for an additional 24 hours in DMEM with 10% FBS in the presence of TGF-β1 (10 ng/mL, PeproTech). In another group of experiments, cells were preincubated with AKT specific inhibitor LY294002 (40 μM, Selleckchem), STAT3 specific inhibitor S3I-201 (50 μM, Selleckchem), P38 inhibitor SB 203580 (10 μM, Selleckchem), or SMAD3 inhibitor SIS3 (3 μM, Selleckchem) for 30 minutes before GSK1016790A treatment.

TRPV4 agonist GSK1016790A (GSK) was purchased from Selleckchem (Houston, TX, Cat. #S6637). TRPV4 antagonist HC067047 (HC) was purchased from Sigma (Saint Louis, MO; Cat. # SML0143). Because TRPV4 can be activated by several stimuli, including heat and mechanical stress (Nilius et al., 2004 (link)), we avoided temperature changes and fast superfusion to study the separate effects of the chemicals. Thus, experiments were conducted at room temperature (22°C–24°C); agonist and antagonist were diluted in external solution and gently dripped onto the recording chamber for final concentrations of 10 nM GSK1016790A and 10 µM HC067047. For TRPV4 inhibition, cells were preincubated with HC067047 at room temperature for at least 30 min before the start of recordings.