Notch3 Modulates Cardiac Fibroblast Proliferation,Apoptosis,and Fibroblast To Myofibroblast Transition Via Negative Regulation Of The RhoA/ROCK/Hif1α Axis

Background: Cardiac fibrosis is a common pathological process in multiple cardiovascular diseases.Abnormal cardiac fibroblast(CF)activity is a key event in cardiac fibrosis.While the Notch signaling pathway has been reported to play a vital role in protection from cardiac fibrosis,the exact mechanisms underlying cardiac fibrosis and protection from it have not yet been elucidated.Similarly,Hif1α and the RhoA/ROCK signaling pathway have been shown to participate in cardiac fibrosis.The RhoA/ROCK signaling pathway has been reported to be an upstream pathway of Hif1α in several pathophysiological processes.In the present study,we aimed to determine the effects of Notch3 on CF proliferation,apoptosis and fibroblast to myofibroblast transition and its relationship with the RhoA/ROCK/Hif1α signaling pathway.Method: The experiment consists of three parts.Part Ⅰ: To clarify the effect of Notch3 on its proliferation,phenotypic transformation,and apoptosis.We applied Notch3 overexpression plasmid and small interfering RNA to overexpress and knockdown Notch3 in CFs which was precultured with fetal bovine serum.Cells were divided into these groups: control group,sc notch3 group,si notch3 group,vector group and ov-N3 ICD group.RT-qPCR and western blot analysis were used to detect the transfection efficiency.CF proliferation was tested by CCK8 and Edu assay.The apoptotic rate was analyzed by flow cytometry.Western blot analysis was applied to observe apoptosis-related proteins such as total caspase3,cleaved caspase3 and Bcl2;and phenotypic transformation-related indicators such as Col I,Col III,and α-SMA.Part Ⅱ: To investigate the relationship between Notch3 and RhoA/ROCK / Hif1α axis.Cells were divided into these groups: NC group,2-ME + NC group,si notch3 group,2-ME + si notch3 group;Vector group,DMOG + vector group,ov-N3 ICD group,DMOG + ov-N3 ICD group;NC group,Y-27632 + NC group,si notch3 group,Y-27632+ si notch3 group.Western blot was used to measure the expression of RhoA,ROCK1,ROCK2,Hif1α,cell membrane,and cytoplasm RhoA.After pretreatment with 2-ME(inhibitor of Hif1α)and DMOG(inhibitor of Proline hydroxylase),the proliferation,apoptotic ratio,apoptosis-related proteins,and cell phenotype conversion-related indicators of cardiac fibroblast were measured.To explore the relationship between Hif1α and the RhoA/ROCK pathway,the CF was pretreated with Y-27632(RhoA/ROCK pathway inhibitor).Part Ⅲ: animal experiment.Notch3 was overexpressed in the myocardium by Ad-N3 ICD injection.The experiment was divided into three groups: sham group,Ad-GFP+MI group,Ad-N3ICD+MI group.The efficiency of Ad-N3 ICD injection was detected by RT-qPCR and western blot.Cardiac function was evaluated by echocardiography.Masson staining was used to detect cardiac fibrosis.Western blot was applied to analyze the expression of α-SMA,RhoA,ROCK1,ROCK2,Hif1α,and cell membrane and cytoplasm RhoA.Results: The results of our research are divided into 3 parts.Part Ⅰ: Notch3 in CFs was overexpressed and knocked down successfully after ov-N3 ICD plasmid and siRNA transfection.Compared with the control group and vector group,Notch3 overexpression inhibited the CF proliferation and differentiation into myofibroblasts,and promoted its apoptosis.Meanwhile,Notch3 overexpression increased the ratio of ccaspase3/t-caspase3 while reduced Bcl2 expression.The Notch3 knockdown had the exact opposite effect.Part Ⅱ: Compared with the control and vector group,Notch3 overexpression reduced the levels of Hif1α,RhoA,ROCK1,and ROCK2,while Notch3 knockdown increased Hif1α,RhoA,ROCK1,and ROCK2.Pretreated with 2-ME before siRNA transfection reversed cell proliferation and the upregulation of Col I,Col III,and α-SMA,which induced by Notch3 knockdown.Pretreatment with DMOG before ov-N3 ICD plasmid transfection reversed apoptosis and apoptosis-related protein changes caused by Notch3 overexpression.Pretreatment with Y-27632 before siRNA transfection abolished the increase of Hif1α caused by Notch3 knockdown.Part Ⅲ: Compared with the sham group and Ad-GFP group,the expression of Notch3 in the myocardium was up-regulated after Ad-N3 ICD injection.Echocardiography revealed that the Ad-N3ICD+MI group had higher LVEF and lower LVEDD than Ad-GFP +MI group.Masson staining showed that the myocardial fibrosis area was smaller in the Ad-N3ICD+MI group than in the Ad-GFP+MI group.Western blot demonstrated that the expressions of α-SMA,Hif1α,RhoA,ROCK1,and ROCK2 in the AdN3ICD+MI group were lower than those in the Ad-GFP+MI group.Meanwhile,the expression of membrane-bound RhoA also decreased in the Ad-N3ICD+MI group.Conclusions: Notch3 switches CF proliferation,apoptosis,and fibroblast to myofibroblast transition;Notch3 regulates cardiac fibroblast activity by inhibiting the RhoA/ROCK/Hif1α axis;Notch3 alleviates myocardial infarction induced cardiac fibrosis through inhibiting RhoA/ROCK /Hif1α axis.