[The mechanism of Rap1GAP in ventricular hypertrophy and interstitial fibrosis induced by angiotensin Ⅱ].

Objective: To investigate the role of Rap1GAP in myocardial hypertrophy and fibrosis and its potential mechanism. Methods: In animal experiments, 20 cardiac-specific Rap1GAP knockout mice (Rap1GAPΔ/Δ) and 20 Rap1GAP homozygous floxed mice (Rap1GAPfl/fl), aged 6-8 weeks, were divided into four groups: Rap1GAPfl/fl+saline group, Rap1GAPΔ/Δ+saline group, Rap1GAPfl/fl+AngⅡ group, and Rap1GAPΔ/Δ+AngⅡ group. Echocardiography was used to assess cardiac function in each group. HE staining was performed to observe the overall structure and cellular morphology of myocardial tissue. Wheat germ agglutinin staining was used to measure the cross-sectional area of cardiomyocytes. Masson staining and Sirius red staining were employed for quantitative analysis of myocardial fibrosis levels and collagen proportion. Western blot was used to detect the protein expression levels of Rap1GAP, α-smooth muscle actin (α-SMA), atrial natriuretic peptide (ANP), β-myosin heavy chain (β-MHC), collagen type Ⅰ, collagen type Ⅲ, transforming growth factor-β1 (TGF-β1), phosphorylated extracellular regulated kinase (p-ERK), extracellular regulated kinase (ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), c-Jun N-terminal kinase (JNK), phosphorylated p38 mitogen-activated protein kinase (p-p38), p38 mitogen-activated protein kinase (p38), phosphorylated nuclear factor κB (p-NF-κB), and nuclear factor κB (NF-κB) in myocardial tissue. In cell experiments, primary cardiomyocytes and cardiac fibroblasts were isolated from 1-3-day-old Wistar rat neonates. Rap1GAP expression was knocked down by transfecting Rap1GAP small interfering RNA (si-Rap1GAP), and cells were divided into si-control (negative control siRNA)+saline group, si-Rap1GAP+saline group, si-control+AngⅡ group, and si-Rap1GAP+AngⅡ group. Rap1GAP was overexpressed by infecting with Rap1GAP adenovirus (Ad-Rap1GAP), and cells were divided into Ad-GFP (GFP empty vector adenovirus)+saline group, Ad-Rap1GAP+saline group, Ad-GFP+AngⅡ group, and Ad-Rap1GAP+AngⅡ group. For inhibitor rescue experiments, cardiac fibroblasts infected with Ad-Rap1GAP or Ad-GFP were treated with 10 μmol/L p38 inhibitor (SB203580) or TGF-β1 inhibitor (pirfenidone), forming Ad-Rap1GAP+AngⅡ+SB203580 group, Ad-Rap1GAP+Ang+pirfenidone group, Ad-GFP+AngⅡ+SB203580 group and Ad-GFP+Ang+pirfenidone group. Immunofluorescence staining was used to detect the expression levels of Rap1GAP, α-SMA, and proliferating cell nuclear antigen (PCNA) in cells. Dihydroethidium staining was employed to measure reactive oxygen species (ROS) levels. Western blot was used to detect the expression levels of target proteins (consistent with animal experiments). Results: In animal experiments, compared with the Rap1GAPfl/fl+AngⅡ group, the Rap1GAPΔ/Δ+AngⅡ group showed larger left ventricular end-diastolic diameter and left ventricular end-systolic diameter, while the cardiomyocyte surface area, myocardial fibrosis ratio and myocardial collagen volume ratio were smaller. Additionally, the expression levels of ANP, β-MHC, collagen type Ⅰ, collagen type Ⅲ, and α-SMA were lower (all P<0.05). Proteomic analysis revealed differences in the protein expression profiles of myocardial tissue between the Rap1GAPΔ/Δ+AngⅡ group and the Rap1GAPfl/fl+AngⅡ group at biological process, cellular component, and molecular function. In cell experiments, compared with the si-Rap1GAP+AngⅡ group, the si-control+AngⅡ group exhibited larger cardiomyocyte cross-sectional area, as well as higher expression levels of ANP, β-MHC, collagen type Ⅰ, collagen type Ⅲ and α-SMA, and a higher proportion of PCNA-positive cells (all P<0.05). Compared with the Ad-GFP+AngⅡ group, the Ad-Rap1GAP+AngⅡ group showed larger cardiomyocyte cross-sectional area, higher expression levels of the aforementioned proteins, and a higher proportion of PCNA-positive cells (all P<0.05). Furthermore, the relative expression levels of p-ERK/ERK, p-JNK/JNK, p-p38/p38, p-NF-κB/NF-κB proteins, and ROS levels in cardiomyocytes or cardiac fibroblasts were higher in the si-control+AngⅡ group than in the si-Rap1GAP+AngⅡ group (all P<0.05), while these indices were higher in the Ad-Rap1GAP+AngⅡ group than in the Ad-GFP+AngⅡ group (all P<0.05). Meanwhile, the ROS level, proportion of PCNA-positive cells, and expression levels of TGF-β1, p-ERK/ERK, p-JNK/JNK, p-p38/p38, collagen type Ⅰ, collagen type Ⅲ, and α-SMA proteins in cardiac fibroblasts were lower in the Ad-GFP+Ang+SB203580 group than in the Ad-GFP+AngⅡ group (all P<0.05). The same trend was observed in the Ad-GFP+AngⅡ+pirfenidone group, with all aforementioned indices lower than those in the Ad-GFP+AngⅡ group (all P<0.05). Conclusions: Rap1GAP may mediate cardiomyocyte hypertrophy by regulating the mitogen-activated protein kinase signaling pathway and the expression of its downstream target NF-κB. Meanwhile, Rap1GAP may promote myocardial fibrosis by inducing ROS production and activating the TGF-β1/mitogen-activated protein kinase signaling pathway.