Study Of The Effect Of P38MAPK Gene Silence On Aldosterone Mediated Cardiac Remodeling

Background Large number of experimental results and clinical evidence confirm that aldosterone can aggravate myocardial ischemia and injury, induce cardiomyocyte apoptosis and necrosis, promote myocardial and vascular extracellular matrix deposition and endothelial dysfunction. Aldosterone levels may accelerate ventricular remodeling and heart failure after coronary artery disease and myocardial infarction (MI), which is the independent risk factor of affecting the development and outcome of cardiovascular disease.ventricular remodeling and heart failure caused by aldosterone and MI have become one of the hotspot and key cardiovascular research, but the mechanisms of aldosterone-inducing ventricular remodeling post MI remain unclear. Aldosterone-inducing ventricular remodeling post MI is closely related to P38MAPK signal pathway. P38MAPK inhibitors can prevent aldosterone-inducing myocardial apoptosis, myocardial hypertrophy, myocardial fibrosis and improve cardiac dysfunction post myocardial infarction and heart pathological remodeling. However, small dose of P38MAPK inhibitors are less effective while high concentrations may inhibit other kinases. P38MAPK inhibitor only block ligands and receptors, but not entirely reveal the gene mechanism. RNA interference (RNA i) technology is emerging as an effective means of gene silencing which can be efficiently and specifically inhibit the expression of genes. So in this study, we further explore the effects and mechanism on aldosterone overload ventricular remodeling after myocardial infarction by P38MAPK gene silencing.Objective To investigate the effect of Lentiviral-mediated P38MAPK RNAi on cardiac remodeling of myocardial infarction in aldosterone overload rats; further explore its genetic, molecular and overall mechanism.Methods Part one Rattus norvegicus mRNA for P38MAP kinase was found in GeneBank and three short hair RNA (shRNA) were designed by software. The pGPU6/GFP/Neo-shRNA plasmid(pGsh) were constructed after the shRNA template annealing and pGPU6/GFP/Neo vector linearized. The pGPU6/GFP/Neo-shRNA plasmid was sequenced and identified. In vitro model, Ang Ⅱ (1μuM) was used for24h in cultured cardiac H9C-2cells. Experiments were divided into six groups, including control group, AngⅡ group, AngⅡ+pGshl group, AngⅡ+pGsh2group, AngII+pGsh3group, AngⅡ+pGsh negtive group. Transfection efficiency was characterized by immunofluorescence. P38MAPKmRNA levels were determined by quantitative real- time PCR. The levels of phosphorylated P38MAPK protein in H9C-2cells were determined by western blot analysis. The P38MAPK-shRNA lentiviral vectors (PGLV-shRNA) were constructed and sequenced. Part two To confirm the effect of aldosterone on cardiac apoptosis, we used an in vitro model with aldo (10nM) in cultured cardiac H9C-2cells. Experiments were divided into six groups, including control group, aldo group, aldo+PGLV-shRNAl group, aldo+PGLV-shRNA2group, aldo+PGLV-shRNA3group, aldo+PGLV-shRNA negtive group. Apoptosis was characterized by TUNEL. P38MAPKmRNA levels were determined by quantitative real-time PCR. The levels of phosphorylated P38MAPK protein were determined by western blot analysis. In vitro model, TGF-β(4nM) was used for24h in cultured cardiac H9C-2cells. Experiments were divided into four groups, including control group, TGF-β group, TGF-β+PGLV-shRNA3group, TGF-β+PGLV-shRNA negtive group. The levels of P38MAPK, CTGF mRNA were determined by quantitative real-time PCR. The levels of CTGF protein, phosphorylated P38MAPK protein and a-SMA protein were determined by western blot analysis.Part three The male SD rat weight ranged from250-300g were included. The rat MI model was performed by ligation of the left anterior descending coronary artery. Rats received normal feed or feed containing aldosterone (dose,1mg/kg/day). PGLV-shRNA (2×108IU/week) was injected through tail vein from1week post-operation to4week post-operation. Experiments were divided into three groups, including Sham group, PGLV-shRNA3group, PGLV-shRNA negtive group.1week and4weeks after operation, cardiac function was examined by echocardiography. Apoptosis was characterized by TUNEL. Myocardial fibrosis was measured by Masson staining. The levels of P38MAPK, CTGF mRNA were determined by quantitative real-time PCR. The levels of CTGF and a-SMA protein were determined by western blot analysis.Results It showed that the the distribution of GFP-tagged pGPU6/GFP/Neo-shRNA(pGsh) mediated by liposome were taken up into H9C-2myocardial cytoplasm and nuclei. The transfection efficiency was70%. It demonstrated that Ang Ⅱ (1μM) significantly increased P38MAPK mRNA expression. Quantitative real-time PCR and western blot demonstrated that pGsh1、 pGsh2and pGsh3markedly decreased the induction of P38MAPK mRNA and phosphorylation P38MAPK protein expression induced by Ang Ⅱ (P<0.05, P<0.05, P<0.01), especially in shRNA3group (P<0.01). The P38MAPK-shRNA with U6promoter lentiviral vectors (PGLV-shRNA) were constructed and sequenced. After stimulation with aldosterone (10nM) for24h, H9C-2 cells showed increasing myocyte apoptosis, P38MAPK mRNA and phosphorylation P38MAPK protein expression compared to those without aldosterone stimulation. It demonstrated that PGLV-shRNA2and PGLV-shRNA3markedly decreased myocyte apoptosis, P38MAPK mRNA and phosphorylation P38MAPK protein expression induced by aldosterone (P<0.05, P<0.01). It demonstrated that TGF-β significantly increased P38MAPK,CTGF mRNA expression(P<0.01, P<0.01) and phosphorylation P38MAPK protein, CTGF protein and a-SMA protein expression(P<0.01, P<0.01, P<0.01). It demonstrated that PGLV-shRNA markedly decreased P38MAPK, CTGF mRNA expression and phosphorylation P38MAPK protein, CTGF protein and a-SMA protein expression induced by TGF-β (P all<0.01). After1weeks post-MI, the echocardiography results showed that aldosterone overload rats have lower FS and LVEF (P<0.01, P<0.01) and enlarger LVEDD, LVESD (P<0.01, P<0.01) compared to those in sham rats. It showed that the the distribution of GFP-tagged PGLV-shRNA was taken up into myocardial cytoplasm for7days. After4weeks post-MI, compared to those in PGLV-NC rats, PGLV-shRNA markedly increased FS and LVEF (P<0.05, P<0.05) and decreased LVEDD,LVESD (P<0.05, P<0.05).It demonstrated that PGLV-shRNA markedly decreased myocyte apoptosis, P38MAPK, CTGF mRNA expression and CTGF protein and a-SMA protein expression in aldosterone overload post-MI rats (P all<0.01).Conclusion In vitro and in rat MI model, aldosterone can directly induce cardiomyocyte apoptosis through P38MAPK pathway. Lentiviral-mediated P38MAPK RNAi can efficiently suppress aldosterone-induced myocardial apoptosis. TGF-P directly induces cardiac fibroblasts of connective tissue growth factor expression through P38MAPK pathway. Lentiviral-mediated P38MAPK RNAi can effectively inhibit TGF-β-induced myocardial fibrosis. This sudy confirms that aldosterone overload decreased cardiac function after myocardial infarction and cardiac remodeling, which is closely associated with the P38MAPK signaling pathway. P38MAPK gene silencing can improve cardiac function and cardiac remodeling after myocardial infarction in aldosterone overload rats.