The Mechanism Of Endogenous Myocardial Protection For Notch1Signaling Pathway

Chapter1IntroductionIt is hypothesized that Notch1plays an important role in myocardial protection as an endogenous cardioprotective factor by cross talking with PI3K/Akt, JAK/Stat3signaling. By technologies including lentiviral vector construction, transgenic cell and rat are established based on Notch1signaling and regard PI3K/Akt, JAK/Stat3as entry point. After confirming the effect of ischemic preconditioning (IPC) and ischemic postconditioning (IPost) in these experimental models, the cardioprotection of Notch1signaling will observed from the cell viability and apoptosis. After determination of mitochondrial membrane potential (△ψm) and reactive oxygen species (ROS), the cytC content will compared in cytoplasm and mitochondria, the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-xl will detected, which will illustrate the molecular mechanism of the Notch signaling for inhibition of myocardial apoptosis. The effect of Notchl expression level on the Akt and Stat3activity will observed to examine the interactions between Notchl, PI3K/Akt and JAK/Stat3signaling. Finally, the cardioprotection of the Notchl signaling will further examed in the isolated rat heart model. This study will clarify the mechanism and significance of Notchl signaling in myocardial protection, and provide a theoretical basis for the clinical treatment of ischemic heart disease.Chapter2Construction of the rat N1ICD lentiviral overexpression and interference vectorsObjective:Construct the high titers rat N1ICD lentiviral overexpression vector (LV-N1ICD) and N1ICD lentivirus interference vector (LV-N1ICD-shRNA), which provide an experimental basis for explore the cardioprotection of Notchl signaling pathway.Methods:With the rat cDNA as a template, the N1ICD fragment was amplified by PCR, which construct pGC-FU-N1ICD-3Flag shuttle plasmid by directly clone.4pairs of N1ICD-shRNA oligonucleotide sequences were synthesized to construct the GVC112-N1ICD-shRNA interference plasmid. pGC-FU-N1ICD-3Flag and GVC112-N1ICD-shRNA plasmids were co-transfected into293T cells to identified the the best interference plasmid in4of GVC112-N1ICD-shRNA by detection Flag expression. Then pGC-FU-N1ICD-3Flag or GVC112-N1ICD-shRNA plasmid togethers with pHelper1.0, pHelper2.0plasmid co-transfect into293T cells to package LV-N1ICD and LV-N1ICD-shRNA, virus titer are determined by Real-time PCR and drug screening method respectively.Results:pGC-FU-N1ICD-3Flag and GVC112-N1ICD-shRNA plasmid are verified by PCR, gene sequencing and Western-blotting, which togethers with pHelper1.0, pHelper2.0plasmid co-transfect into293T cells can obtain the high titer LV-N1ICD and LV-N1ICD-shRNA by concentrated the cell supernatant.Conclusions:The LV-N1ICD and LV-N1ICD-shRNA are successfully constructed and t packaged, which have the Notchl signaling biological functions. Chapter3Notch1exert endogenous card ioprotect ion by crosstalk with PI3K/Akt, JAK/Stat3signal pathwayObjective:To defined Notch1can attenuate myocardial ischemia-reperfusion injury (IRI), and explore the endogenous cardioprotective mechanism of the Notch1signaling.Methods:The N1ICD/Hesl protein expression are detected after the myocardial hypoxia, hypoxia/reoxygenation (H/R), IPC, IPost model were established in vitro. H9c2cells are suffer from H/R, IPC and IPost treatment following infected with LV-N1ICD and LV-N1ICD-shRNA respectively, CCK-8was used to assess cell viability, flow cytometry was used to detect apoptosis,△ψm and ROS. Segregate mitochondria from H9c2cell to compare the cytC expression in the cytoplasm and mitochondria. The expression of Bax, Bcl-xl, PTEN, p-Akt/Akt、 p-GSK-3β/GSK-3β and p-Stat3/Stat3were detected by western-blotting.Results:The Notchl signaling is activated in myocardial ischemic injury. The force expression of N1ICD during myocardial H/R can improve cell viability, inhibit apoptosis, stable△ψm, reduce ROS formation and decrease mitochondrial cytC release to the cytoplasm. The activated Notchl signaling in H/R can down-regulat the expression of PTEN, Bax and up-regulated the expression of Bcl-xl, p-Akt, p-GSK-3β, p-Stat3, simultaneously. However, inhibition of Notchl signaling would weaken Akt and Stat3activity, which abolish the cardioprotective role of IPC and IPost.Conclusions:As an endogenous cardioprotective factor, Notchl signaling can reduce myocardial IRI by cross talking with PI3K/Akt, JAK/Stat3signaling, which would regulate apoptosis-related protein expression and inhibit mitochondrial permeability transition pore opening. Chapter4Notch1signaling improve the cardiac function after myocardial ischemiaObjective:Explore the improvement role of Notch1signaling in cardiac function after myocardial ischemia.Methods:3weeks after LV-N1ICD and LV-N1ICD-shRNA infected respectively, Ischemia/reperfusion (I/R), IPC, IPost model were created using Sprague Dawley rats by the Langendorff isolated heart perfusion, the each period of hemodynamic parameters were real-time observated by PowerLab system, the lactate dehydrogenase (LDH) was detected at lmin before ischemia,10min after reperfusion,2h after reperfusion, the myocardial infarct size (IS) was finally determinated by TTC staining.Results: The Sprague Dawley rats I/R, IPC, IPost model were successfully created by the Langendorff isolated heart perfusion. Activation of Notchl signaling in myocardial I/R can improve the hemodynamic parameters, decrease LDH release and reduce IS, whose cardiac function have no significant difference compared with myocardial IPC, IPost.Conclusions:The Notchl signal can improve heart function after myocardial ischemia.