Effect Of Activation Of Notch-1on Myocardial Ischemia/Reperfusion Injury And Expression Of Bcl-2,Akt,ERK1/2

BackgroundAs one of the most common non-infectious chronic diseases, coronary heart disease (CHD) has been seriously jeopardizing health and quality of life of human beings. The pathogenesis of CHD, However, is not thoroughly identified. It is of much importance to study the relevant risk factors of CHD.Acute myocardial infarction(AMI) leads to high motality rate, cardiac dysfunction, poor quality of life, damage to human health and burden to families and society. During the past decades, a tremendous development has been performed in the diagnosis and therapy of AMI. Percutaneous Coronary Intervention (PCI) and Coronary Artery Bypass Grafting (CABG) improve the prognosis of AMI. Howevery, the side-effect such as myocardial ischemia reperfusion injury (MIRI) decreases therapeutic effect.MIRI is a pathological condition characterized by an initial restriction of blood supply to an organ followed by the subsequent restoration of perfusion and concomitant reoxygenation. In its classic manifestation, occlusion of the arterial blood supply is caused by an embolus and results in a severe imbalance of metabolic supply and demand, causing tissue hypoxia. Perhaps surprisingly, restorationof blood flow and reoxygenation is frequently associated with an exacerbation of tissue injury and a profound inflammatory response, as called reperfusion injury. A wide range of pathological processes contribute to ischemia and reperfusion associated tissue injury. For example, ischemia and reperfusion leads to the activation of cell death programs, including apoptosis (nuclear fragmentation, plasma membrane blebbing, cell shrinkage and loss of mitochondrial membrane potential and integrity), autophagy-associated cell death (cytoplasmic vacuolization, loss of organelles and accumulation of vacuoles with membrane whorls) and necrosis (progressive cell and organelle swelling, plasma membrane rupture and leakage of proteases and lysosomes into the extracellular compartment). MIRI is still an unsolved problem of mycardial ischemia for long time, although many new drugs and methods have been used to treat MIRI, there is no systemitic procedure to cure it since most of the methods are ectogenic.Notch signalling is an evolutionarily conserved intercellular pathway controlling cell differentiation, cell fate specification and patterning during embryonic and postnatal development. In mammals, the pathway encompasses five transmembrane ligands:D11-1(Delta-like1), Dll-3, Dll-4, Jagl (Jagged-1) and Jag2, and four single-pass transmembrane receptors, Notchl-Notch4. Activation of Notch by its ligands initiates a series of successive proteolytic cleavages. First, extracellular cleavage of Notch occurs by TACE [TNF a (tumour necrosis factor α)-converting enzyme; Also called ADAM (a disintegrin and metalloproteinase)17] and Kuzbanian (ADAM10), two members of the ADAM family. This is followed by transmembrane cleavage by γ-secretase, releasing the NICD (Notch intracellular domain), which next translocates to the nucleus and forms a complex with the transcriptional regulator RBP-J κ (recombinant signal-binding protein1for J κ). In the absence of Notch signalling, RBPJ κ associates with transcriptional co-repressors that actively keep target gene expression shut down. Binding of NICD to RBP-J κ replaces the co-repressor transcriptional complex with a co-activator complex, which in turn triggers the transcription of Notch target genes such as bHLH (basic helix-loop-helix) proteins, Hes (hairy/enhancer of split), Hey (Hes-relatedprotein) and Nrarp (Notch-regulated ankyrin repeat protein). These transcription factors regulate further downstream genes to respond to environmental stimuli, either by maintainingcells in an uncommitted state or by inducing differentiation depending on cell typeRecent studies provide evidence that one physiological activity of Notch is a function of its ability to increase survival in many cell types. Several pathways have been shown to contribute to the survival effect of Notch. The "inhibitor of apoptosis proteins"(IAPs) are caspase inhibitors originally identified in baculoviruses for their ability to inhibit apoptosis in host cells. In mammalian cells, IAP family members act by binding to and inhibiting caspases-3,-7, and-9. X-linked inhibitor of apoptosis protein (XIAP) is one of the best-characterized IAP members in terms of both its structureand biochemical mechanism. The regulation of cell survival by Notch intracellular domain could partly be attributed to a selective increase of XIAP. Activated Notch1was found to inhibit p53-induced apoptosis and sustains transformation by human papillomavirus type16E6and E7oncogenes through a PI3K-PKB/Akt-dependent pathway.Notch signal blockade resulted in down-regulation of Hes5, leading to reduced formation of the Hes5-STAT3complex and hypophosphorylation of STAT3, which further attenuated manganese superoxide dismutase (MnSOD) expression and increased ROS and apoptosis in hepatocytes during ischemia/reperfusion injury. Notch-1was found to be activated in border zone Cardiomyocytes after myocardial infarction. Overexpression of activated Notch intracellular domain prompts a3-fold increase in phosphorylated Akt. Infarcted hearts injected with adenoviral vector expressing Notch intracellular domain treatment exhibit improved hemodynamic function, implicating Notch signaling in a cardioprotective role following cardiac injury. The role of Notch-1involved in MIRI has not been established, not to mention the molecular mechanisms.Objectives:1. To determine the expression levels of Notch pathway in animal model of MI and MIRI, respectively;2. To investigate the molecular mechanisms of Notch involved in the protective effect of cardiac myocytes in MIRI.Methods:1. In vitro study54male Sprague Dawley rats were randomly divided into3groups:Sham operation group, myocardial infarction (MI) group, and myocardial ischemia reperfusion (MIR) group. The left anterior descending branch of coronary artery was ligated for30min to make the myocardial infarction model. MIR was induced by ligating the left anterior descending artery (LAD) for30min, followed by loosening the ligature for120min. Successful ligation of LAD was evidenced by immediate regional cyanosis in the anterior ventricular wall and the apex of the heart with color change greater than40%of the left ventricle (LV) and confirmed by electrocardiography(ECG). All animals were injected with5%Evans blue through right jugular vein and then with cardioplegic solution (10%potassium chloride).Hearts were excised and sectioned transversely into five sections, including one section at the site of ligature. Sections were then incubated in1.5%triphenyltetrazolium chloride (TTC). After TTC staining, viable myocardium stains red and the infarcted areas appear pale.The mRNA level of Notch pathway was determined by QRT-PCR and protein level was determined by Western Blotting.2. In vitro study Ventricles were dissected from1-2day-old Sprage Dawley (SD) rats and rapidly excised, rinsed in ice cold Hanks’ balanced salt solution (HBSS) without Ca2+and Mg2+, washed to remove blood and debris. Remove atriums from isolated hearts. Ventricles are left and moved in a bottle which with HBSS. Then ventricles are shredded into tissue fragments (lmm3) and then digested by stepwise trypsin and collagenase II at37℃. Each step needs8min, shake bottle every4min. Use glass pipette to blow the fragments. Aspirate supernatant and move into another tube with10%FBS DMEM to stop digestion. Repeat the steps (4-5step) until the fragments disappear. Centrifuge cell suspension with1000rpm for5min. And get rid of the suspension. Add DMEM (10%FBS) and re-suspend the cells. Filter cells with filter (200mesh). Cells were pooled, preplated for90min on an uncoated dish to remove fibroblasts, and plated on1%gelatin-coated cell culture plastic dishes in Dulbecco’s Modified Eagle’s Medium supplied with0.2%BSA,3mM sodium-pyruvate,0.1mM ascorbic acid,4mg/liter transferrin,2mM L-glutamine, and5mg/liter ciprofloxacin supplemented with10%fetal bovine serum at1x106cells/cm2. After24h, media was changed and cultured until use.Cells were randomly divided into3groups:hypoxia/reoxygenation(H/R) group, H/R+recombinant soluble Jagged-1FC chimera(sJag-1) group, and H/R+DAPT group.To mimic an I/R model (via hypoxia in the medium), cells at80%confluence were incubated with a glucose-free medium (previously bubbled with95%N2and5%of CO2) for3h at37℃in a hypoxic chamber (95%N2and5%CO2). Then the medium was replaced by DMEM and cells were cultured under normal oxygen during the reoxygenation period. Notch-1ICD and Hes-1protein levels were determined by Western Blotting. Cell necrosis and apoptosis rates were mearsured by Flow Cytometer afer staining by Annexin V/PI.Infectious AAV8vector particles were generated in HEK293cells by cotransfecting each vector plasmid:pAAV-N1ICD or pAAV-PEGFP together with the packaging plasmid and helper plasmid, expressing AAV and adenovirus helper functions, respectively. Viral stocks were obtained by CsCl2gradient centrifugation; rAAV titers, determined by measuring the copy number of viral genomes in pooled, dialyzed gradient fractions, were in the range of～1012to～1013genome copies per milliliter. Cells were divided into3groups:control group, cardiac myocytes transfected with pAAV-PEGFP group, and cardiac myocytes transfected with pAAV-N1ICD group. Western Blotting was used to evaluate the expression of p-Akt, p-ERKl/2.Results:1. In vivo study1.1D11-4, Jagged-1, Notch-1, Notch-2, Notch-3, and Notch-4could be detected in normal adult rat myocardium。 Significant changes of Notch pathway were detected during MI and MIRI:(1) As compared with sham group(1.03±0.20), D11-4mRNA were lower in MI group(0.62±0.17) and MIRI group(0.32±.020,F=31.67,P<0.001);(2)As compared with sham group(1.01±0.06), Jagged-1mRNA were higher in MI group(1.21±0.12) and lower in MIRI group(0.42±0.05, F=225.88, P<0.001);(3) As compared with sham group(0.99±0.07), Notch-1mRNA were higer in MI group(2.40±0.11) and lower in MIRI group(0.31±0.05, F=1610.03, P<0.001);(4) As compared with sham group(1.01±0.02), Notch-2mRNA were lower in MI group(0.71±0.02) and MIRI group(0.62±0.02, F=1083.85, P<0.001);(5) As compared with sham group(0.99±0.04), Notch-3mRNA were lower in MI group(0.92±0.02) and MIRI group(0.56±0.02, F=488.65, P<0.001);(6) As compared with sham group(1.02±0.10), Notch-4mRNA were lower in MI group(0.80±0.10) and MIRI group(0.52±0.12, F=48.08, P<0.001). 1.2Notch-1intracellular domain(Notch-1ICD) and its target gene Hes-1could be detected in normal adult rat myocardium。 Significant changes of Notch pathway were detected during MI and MIRI:(1) As compared with sham group(1.20±0.09), Notch-1ICD were higher in MI group(1.62±0.07) and lower in MIRI group(0.14±0.08, F=853.39, P<0.001);(2) As compared with sham group(1.72±0.19), Hes-1were higher in MI group(3.90±0.22) and lower in MIRI group(0.49±0.17, F=723.00, P<0.001).2. In vitro study(1) The apoptosis rate of cardiomyocytes in H/R group(0.742±0.021) was significant higher than that in normal oxygen group(0.045±0.010) and hypoxia group(0.432±0.026, F=2727.43, P<0.001). The hypoxia/reoxygenation model was successful.(2) The apoptosis rate of Cardiomyocytes in H/R±sJag-1group(0.490±0.047) was significant lower than that of H/R group(0.585±0.097) and H/R±DAPT group(0.778±0.064, P<0.001, F=23.985, P<0.001)(3) pAAV-N1ICD virus was constructed successfully with titre of2X1013pfu/ml was amplified by HEK293cells and collected.(4) As compared with control group(0.10±0.01), p-Akt were higher in pAAV-N1ICD transfection group(0.51±0.02, F=1368.472, P<0.001);(5) As compared with control group(1.20±0.09), p-ERK1/2were higher in pAAV-N1ICD transfection group(ERK1, x2=22.056, P<0.001; ERK2, x2=19.474,P<0.001);(6) As compared with control group(0.09±0.02), Bc1-2were higher in pAAV-N1ICD transfection group(0.38±0.04, F=354.774, P<0.001).Conclusions 1. Rat MI and MIRI models were constructed, Jagged-1and its receptor Notch-1up-regulated in MI and down-regulated in MIRI;2. Up-regulation of Notch-1ICD by Notch-1receptor ligand sJag-1had protective effect on neonatal cardiac myocytes under hypoxia/reoxygenation.3. Notch-1ICD gene was effectively transfected into neonatal cardiac myocytes. N1ICD transfection had significant protective effect on hypoxia/reoxygenation. The mechanisms might be suppression of cell death by activation of p-Akt, p-ERKl/2, and Bcl-2.