Animals and Animal Models

Male C57BL/6 mice aged 9–10 weeks with a weight of 24.5–29.5 g were housed in the pathogen-free mouse room. The mice were subjected to chronic angiotensin II (1.0 mg/kg/ day) via osmotic minipumps to establish a cardiac fibrosis model. P-Rex1 expression in the whole heart tissue was examined on days 3, 7, 14, and 28 (n = 6 for each group). In addition, angiotensin II-induced cardiac remodeling mice received daily intraperitoneal injections of 50 μl PBS, 5 mg/kg mouse 1A-116 (Cat. No. 6701/10, from Tocris company), which is a P-Rex1 specific inhibitor and has been proved by Cardama (n = 6 for each group). At 4 weeks, the hearts were dissected and weighed to calculate the heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) ratios. All the experimental procedures were performed following the institutional guidelines of animal care and were approved by the ethics committee of the first affiliated hospital of USTC University.

Culture of Cardiac Fibroblasts

The hearts were taken from one to three days old neonatal rats and cardiac fibroblasts (CFs) were isolated. After 24–48 hours of growth, CFs would be passaged in a ratio of 1:3 and used for the indicated experiments. CFs were assigned to different groups, including PBS group, AngII group, siRNA group, 1A-116 treatment group, AngII+1A-116 group, AngII+siRNA group, respectively. CFs were incubated with angiotensin II human (10–5 M, Sigma, USA) for the indicated time.

Active Rac1 Pull-Down

Approximately, 70–80% of the confluent of NRCFs was lysed with lysis buffer and then the pellet was collected after centrifuge (14,000×g for 5 min at 4 °C). For the detection of active Rac1-GTP, the Rac1 Activation Magnetic Beads Pull-down Assay (Merck, 17-10393, Darmstadt, Germany) was applied according to the manufacturer’s instructions. Then, the samples were applied to a polyacrylamide gel along with the beads and transferred onto membranes. First, we use ponceau S in 0.1% acetic acid stain for total protein normalization. Then, we washed ponceau S out and the membranes were blocked and then incubated with anti-Rac1 (ab282581, Abcam, 1:1000) antibodies at 4 °C overnight. The secondary antibodies were incubated at room temperature for 1 h. The blots were visualized using a High-sig ECL western blotting substrate (Tanon, Shanghai, China).

Western Blot Analysis

The whole heart tissue or CFs was lysed in RIPA lysis buffer, and the total protein was extracted and detected with a BCA Protein Assay Kit. Approximately 20 μg of total protein was separated by electrophoresis on 6% to 15% SDS-polyacrylamide gels. After electrophoresis, the samples were transferred to immobilon-FL PVDF membranes. The membranes were blocked and then incubated with anti-P-Rex1 (ab175431, Abcam, 1:1000), anti-Rac1 (ab282581, Abcam, 1:1000), anti-CTGF (sc-365970, Santa Cruz, 1:800), anti-α-SMA (sc-53142, Santa Cruz, 1:1000), anti-collagen1 (sc-59954, Santa Cruz, 1:500), anti-Paks (2604, Cell signaling, 1:1000), anti-phospho-Paks (2606, Cell signaling, 1:1000), anti-total ERK1/2 (4695, Cell signaling, 1:1000), anti-phospho-ERK1/2 (4370, Cell signaling, 1:1000), anti-P38 (9212, Cell signaling, 1:1000), anti-phospho-P38 (4511, Cell signaling, 1:1000), and anti-GAPDH (5174, Cell signaling, 1:1000) antibodies at 4 °C overnight. The secondary antibodies were incubated at room temperature for 1 h. The blots were visualized using High-sig ECL western blotting substrate (Tanon, Shanghai, China).

Migration and Proliferation Assay

CFs were seeded in a 24-well plate or cultured in the Transwell unit. CFs were starved overnight and cultured for 24 h with inhibitor or siRNA for P-rex1, then stimulated with AngII. The fibroblasts would be incubated with Roti©-ImmunoBlock at RT, the Ki67 antibody (12075, Cell signaling, 1:200) was added overnight at 4 °C. Then the fibroblasts were washed thrice with PBS and incubated with the respective secondary antibody, DAPI, and TRITC-phalloidin for 1 h at RT. Images were taken with a fluorescence microscope to count the ratio of Ki67-positive cells to DAPI-stained nuclei. The invasive cells were counted as the number of migrated fibroblasts under Zeiss microscope.

Measurement of Nox Activity

The Nox activity in the CFs was measured by enhanced lucigenin chemiluminescence. The debris was removed by centrifugation at 12,000×g for 10 min at 4 °C and the supernatant was collected; 100 μM of NADPH was mixed with the supernatant and reacted with Nox to generate superoxide anions. The reaction of lucigenin (5 μM) with superoxide anions showed light emission, and the values represented the Nox activity as the mean light units (MLU) per minute per milligram of protein.

RNA Isolation and Real-Time RT-PCR

The total mRNA was extracted from whole cardiac tissues using TRIZOL reagent, and cDNA was synthesized according to the manufacturer’s instructions. The primer sequences were for P-Rex1: 5’-GGCATTCCTGCATCGCATC-3’, reverse primer: 5’- CGGGTGTAAACAATACTCCAAGG-3’; α-SMA: 5’-ACTCTCTTCCAGCCATCTTTCA-3’, 5’-ATAGGTGGTTTC GTGGATGC-3’; collagen I: 5’-CATGTTCAGCTTTGTGGACCT-3’, 5’-GCAGCTGACTTCAGGGATGT-3’; CTGF: 5’-TGTGTGATGAGCCCAAGGAC-3’, 5’-AGTTGGCTCGCATCATAGTTG-3’; IL-6:5’-AGTTGCCTTCTTGGGACTGA-3’, 5’-TCCACGATTTCCCAGAGAAC-3’. The relative mRNA expression of P-Rex1, CTGF, collagen 1a1, IL-6, and α-SMA were analyzed by RT-PCR and normalized to the expression of the PPIA housekeeping gene.

Echocardiography

Echocardiographic images were obtained using an ultrasound system with a 21-MHz probe under isoflurane anesthesia (2.0–3.0%). When the heart rate was around 400 bpm, we measured the left ventricle (LV), the heart rate (HR), LV end-systolic diameter (LVESD), LV end-diastolic diameter (LVEDD), LV posterior wall thickness (LVPWD), end-diastolic ventricular septal thickness (IVSD), ejection fraction (EF), and fractional shortening (FS) using two-dimensional parasternal long-axis and short-axis views at the level of the papillary muscle.

Histological Analysis

After hearts were fixed with 4% neutral paraformaldehyde for 7 days, cardiac sections (5 μm) of left ventricular sections were cut from the same location of each heart and examined by Hematoxylin and eosin (H&E) staining and Sirius red staining (for morphometric analysis, photographs were observed under × 400 magnification with Carl Zeiss GmbH, Oberkochen, Germany). Interstitial fibrosis and the cardiac fibrosis volume fraction were calculated as the ratio of the stained fibrotic area to the total myocardial area (the mean percentages of collagen fiber occupied parts in LV (%/mm2 of field)).

Statistical Analyses

Data from three to five independent experiments were shown as the mean±SD and processed using Prism 7 (GraphPad Software Incorporated, USA). One way ANOVA with Dunett-t post hoc correction was used to compared between the individual groups and the control group. We show a representative immunoblot in the main figures, changes in protein/phosphorylation levels from three independent experiments were semi-quantified by densitometry and were shown as ratios or fold changes from different groups. A p-value < 0.05 was considered significant.