Role Of Notch Signaling Pathway In Myocardial Ischemia Reperfusion Injury And The Underlying Molecular Mechanisms

【Background】Ischemia reperfusion injury (IRI) refers to reperfusion could not improve functionalrecovery, but increase dysfunction and structural damage of tissues and organs afterischemia. IRI can occur in myocardial ischemia, stroke, trauma and other diseases andpathological processes. Ischemic heart disease is one of the world’s leading causes ofdeath, which is a serious threat to public health. To save ischemic myocardium, it must benecessary for timely and effective restoration of blood flow to ischemic myocardium.However, clinical findings show that a proportion of patients of ischemic myocardiumlead to myocardial ischemia/reperfusion (myocardial ischemia/reperfusion, MI/R) injuryin spite of successful surgery to restore the blood supply, resulting in worsened condition, appearing progressive decline in cardiac function, and even fatal arrhythmias. There arethree aspects for the impact of MI/R injury: change in myocardial ultrastructure,myocardial energy metabolism, and the cardiac function. The production in reactiveoxygen species (ROS) due to restoring oxygen increases after reperfusion, calciumoverload and inflammation, are mechanisms causing MI/R injury, and directly orindirectly cause myocardial apoptosis and necrosis.Notch signaling pathway, having influence on cell fate, is conserved signaltransduction pathway, which determines the maintainance of normal structure and functionin adult animal tissues and organs and repair of damage after injury. In mammals, forexample, the classical Notch signaling pathway includes five kinds of ligands (Delta-like1,3,4, Jagged1, and Jagged2) and four receptors (Notch1, Notch2, Notch3, Notch4). Whenthe ligand binds the receptor, Notch signal is activated, and under the γ-secretase cleavagethe receptor intracellular domain (Notch intracellular domain, NICD) is cut andtranslocated into the nucleus. It combined to the nuclear transcription factor RBP-J(recombination signal binding protein Jκ), which starts the expression of downstreamgenes of basic helix-loop-helix factor, including the Hes family, triggering a subsequentmolecular events. In recent years, there are reports confirm that Notch signals involve inthe response of cells to external stimuli, participating in the liver and brain ischemic injury.Is Notch signaling involved in MI/R injury? What is the role of Notch signaling in MI/Rinjury? What is the molecular mechanism responsible for the role of Notch signaling inMI/R injury? Clarification of these issues will open up new areas for MI/R injury-relatedresearch, further improve the theoretical system of molecular regulation in MI/R injury,and provide a theoretical basis for the prevention and treatment of MI/R injury.【Aims】To demonstrate the role of Notch signaling pathway in MI/R injury, we observedchanges of Notch signaling pathway-related molecules after hypoxia/reoxygenation, andwhether Notch signaling pathway affected the generation of ROS. The present study is toexplored the molecular mechanism of MI/R injury, and to provide a new strategy and way for the prevention and treatment of ischemic heart disease.【Methods】1. Culture of H9C2(2-1) rat embryonic cardiomyocytes DMEM medium containing100ml/L fetal bovine serum were used. When the cells near confluence, with2.5g/L trypsindigestion, the H9C2(2-1) cells were passaged at1:2to1:4. At1d before the experiment,cell culture was changed into that containing20ml/L fetal bovine serum, leading to H9C2(2-1) cells differentiation into cardiomyocytes.2. Primary culture of neonatal rat cardiomyocytes Under sterile condition, the heart of1~3d neonatal SD rat was remove, and cut into pieces, adding digestive juices containing1.0g/L collagenase Ⅰ for water bath at37℃3~5min. After repeated pipetting andstanding, the supernatant was discarded. After adding the pellet digestion at37℃waterbath for about5min, repeated pipetting, the supernatant was moved to complete mediumwith100ml/L fetal bovine serum and0.1mmol Brdu (containing10%fetal bovineserum100U/ml penicillin and0.1mg/ml streptomycin DMEM). Repeat these steps untilall organizations have been fully digested. Sedimentation centrifugation and resuspend incomplete medium. After differential adhesion90~120min, the cell suspension culturewas moved in plate or dish, adding complete medium containing0.1mmol/L Brdu. Withevery24h for a changing fluid. When changing fluid, cell morphology was observed. Ifthe cell become circular or irregular shaped spindle, and the rest become a regularcontraction, the subsequent experiment was performed.3. Grouping the myocardiocytes were divided into two groups, namelyhypoxia/reoxygenation (H/R) group and the normal group. H/R group used sugar-free andserum-free DMEM culture medium, and was placed in low oxygen tank for6h, thenchanged to normal medium for3h. Normoxic group used high glucose DMEM culturemedium containing100ml/L fetal bovine serum. Each group was subdivided into controlgroup, DMSO group and GSI Group.4. Application of GSI blocking Notch signaling in vitro At day1prior myocardialhypoxia,75μmol/L GSI dissolved in complete medium according to1:3000was added. Blocking effects of Notch signaling pathway was observed for MI/R injury.5. Detection of mRNA expression levels of Notch signaling pathway molecules usingReal-time PCR RNA of primary neonatal rat cardiomyocytes was extracted total usingRNA extraction kit, and reverse transcribed into cDNA using reverse transcription kit. Thereaction system was2×qPCR TaqMix12.5μl,10μmol/L of amplified Notch1, Jagged1,mixtures of forward and reverse primer sequences of genes Hes10.5μl, correspondingcDNA1μl, and water was added to25μl, and negative control without template (2×qPCR TaqMix12.5μl,10μmol/L forward and reverse amplification primer sequencesmixture of Notch1, Jagged1, Hes1gene0.5μl, and water was added to25μl).Fluorescence quantitative PCR amplification was used, and PCR reaction conditions wereas follows: after95℃for5min denaturation,95℃for15s�'60℃for35s(fluorescence detection), and a total of40cycles. PCR products were electrophoresis.6. Western blot for Notch signaling pathway-related molecules Protein was extractedfrom primary cultured neonatal rat cardiomyocytes, protein concentration was determinedby SDS-PAGE electrophoresis and electrically transferred to polyvinylidene fluoride(PVDF) membranes.5%skim milk was used for block for1h.1:500rabbit anti-Notch1,1:200or1:1000Hes1goat anti-mouse anti-β-Tubulin antibody were used and incubatedovernight at4℃, and the membrane was washed with TBST, and then1:4000HPR-labeled goat anti-rabbit, cow goat anti-mouse and goat anti-IgG were used, andincubated at37℃for1h. After washing with enhanced chemiluminescence liquid (ECL),Quantity One analysis software was used for signal acquisition and grayscale scanning.7. Detection of apoptosis H9C2(2-1) cells were seeded onto coverslips. Afterhypoxia/reoxygenation, cells were stained in strict accordance with instructions of TUNELkit, and were stained with DAPI for nuclear staining. Positive criteria was that apoptoticcells were green, indicating positive apoptotic cells, blue was nuclei. The number ofapoptotic cells was observed per field at400times magnification, each slide randomlyselected10vision for cell counting with each count of100vision, and results were givenas the mean. Percentage of apoptotic index (AI) was the ratio of the number of theapoptotic cells to the total number of nucleus (the total number of cells). Staining and cell counts were double-blind.8. Detection of ROS levels using fluorescent probe DCFH-DA Trypsin was used todigest adherent H9C2(2-1) cells. The probe solution was diluted to the desiredconcentration in new DMEM culture medium as a stain solution to replace cell culturefluid, and incubated in the dark at37℃for20min (shaking cells every5min). After theincubation, the cells were washed with fresh DMEM medium, adding0.5~1ml ice-coldPBS to resuspend cells (5~100,000). Using flow cytometry excitation light at488nmwavelength, emission was measured at wavelengths longer than525nm, and the cells canbe divided into two subgroups: ROS-negative with low cell fluorescence intensity andROS-positive cells with strong fluorescence.9. Statistical analysis Data were given as with±s, and origin8software was used forstatistical analysis. ANOVA was used when multiple comparisons were made, andcomparisons between two observations were assessed by t test. P <0.05was consideredstatistically significant.【Results】1. Blocking Notch signaling pathway lead to worse MI/R injury. TUNEL staining wasused to detect the level of apoptosis, and results in vitro showed that H/R significantlyincreased apoptosis rate, application of GSI to block Notch signaling pathway furtherincreased apoptosis rate.2. Blocking Notch signaling pathway increased ROS level in cardiomyocytes sufferedfrom MI/R injury. Fluorescent probe DCFH-DA was used to determine ROS level incardiomyocytes, and results in vitro showed ROS level was significantly increased in H/Rcardiomyocytes. Application of GSI to block Notch signaling pathway further promotedthe generation of ROS in H/R cardiomyocytes.3. Gene expression of each molecule in Notch signaling pathway reactively increased inMI/R injury. H/R significantly elevated levels of Notch1receptor gene in Notch signalingpathway and ligand gene Jagged1levels in cardiomyocytes. After administration of GSI,receptor and ligand Jagged1genes did not change significantly. H/R significantly increased the level of gene of Notch signaling pathway downstream, transcription Hes1, incardiomyocytes. Administration of GSI significantly reduced the level of Hes1gene.4. The expression of protein levels in Notch signaling pathway increased reactively whenMI/R injury. In vitro results showed that H/R significantly increase protein expressionlevels of NICD1and molecule of Notch signaling, the transcription factor Hes1;administration of GSI did not affect protein levels of NICD1, but significantly inhibitedthe protein expression level of Hes1.In summary, in the present study we examined molecules expression levels in the Notchsignaling pathway in H/R cardiomyocytes, and observed expression of Notch signalingmolecules after inhibition of the Notch signaling pathway, suggesting that Notch signalingpathway reactively increased in MI/R, and might be a compensatory protective response.We further detected apoptosis in cardiomyocytes and found that H/R significantlyincreased the level of myocardial apoptosis, and further increased the level of apoptosisafter the Notch signaling pathway was blocked by GSI, indirectly indicating that Notchsignaling pathway may have a protective effect on ischemic myocardium. Further studiesshowed that the level of ROS in cardiomyocytes also significantly increased afteradministration of GSI, suggesting that an increased ROS generation is the mechanism forblocking Notch signaling pathway lead to further MI/R injury.【Conclusion】1. Notch signaling deficiency causes aggravated myocardial ischemia/reperfusion injury,indicating Notch signaling pathway plays an important role in the regulation ofmyocardial ischemia/reperfusion injury. The signal molecule genes and proteins in the I/Rreactivity increases, which might be a compensatory and protective reaction of cells.2. Notch signaling may fight aganist myocardial damage by down-regulating ROS level ofcardiomyocytes in MI/R. Notch signaling blockaye further elevates ROS level, thusincreases cardiomyocyte injury.