Mechanism Of Notch1 Protects Against Myocardial Ischemia-reperfusion Injury Via Regulating Mitochondrial Fusion And Fission

Background and Purpose:Reducing myocardial Ischemia reperfusion injury（I/R）remains an important challenge in modern ischemic heart disease therapeutics.Mitochondria are the power factories of cells,which determine whether cells survive or not.A large number of studies have proved that various signaling pathways of myocardial protection ultimately target mitochondria.The life cycle of mitochondria involves repeated cycle of cleavage and fusion.In order to maintain the normal function of cells,mitochondria maintain a continuous balance in local fusion and cleavage,respectively generating long and narrow continuous or fragmented mitochondria,which can protect cells from Ca²⁺overload,oxidative stress,and Mitochondrial DNA（mtDNA）.mtDNA damage caused by mutations.Mitofusin（MFN）protein is composed of MFN1 and MFN2 of the outer membrane and Optic atrophy protein 1（OPA1）of the inner membrane.Mitochondrial Fission protein consists of the dynamin-related protein 1（DRP1）of cytoplasm and the human mitochondrial Fission protein 1（FIS1）of the outer membrane.In vitro and in vivo studies have shown that mitochondrial fusion is a compensatory mechanism for maintaining normal function and plays an important role in reducing myocardial I/R.Overexpression of DRP1 and FIS1 increased the sensitivity of Mitochondrial permeability transition pore（mPTP）opening,while overexpression of MFN217 or inhibition of DRP118 prevented mPTP opening.Multiple studies have shown that the Notch1 signaling pathway is associated with mitochondrial fusion/cleavage to varying degrees.Notch1 signaling pathway inhibits mitochondrial division and alleviates cell apoptosis through intracellular NICD.In gastric cancer and breast cancer cells,LMP2A can promote mitochondrial cleavage by upregulation of DRP1,while activation of Notch1 signaling pathway breaks the mitochondrial fence-cleavage balance.Therefore,this paper intends to study the mechanism of Notch1 signaling pathway regulating mitochondrial fusion/cleavage to exert myocardial protection,and to prepare the model of ischemia reperfusion injury between myocardial cells and rats by using adenovirus construction and other techniques.On the basis of confirming the occurrence of mitochondrial fusion/cleavage in myocardial and rat ischemia-reperfusion injury models,we investigated the effects of different levels of Notch1 expression on mitochondrial fusion/cleavage proteins Notch1,MFN1,MFN2,OPA1,DRP1,FIS1.The physiological role of Notch1 in regulating fusion/lysis to reduce myocardial I/R was discussed in terms of cell viability and apoptosis.Double luciferase reporter gene,chromatin immunoprecipitation and gel migration techniques were used to investigate the interaction between Notch1,MFN1and DRP1,and to explore the molecular mechanism of Notch1 blocking mitochondrial cleavage and inhibiting mPTP opening.Finally in the whole animal experiment on further comparison,tries to clarify Notch1 signaling pathway to promote mitochondria fusion,inhibiting myocardial protective effect of the molecular mechanism of mitochondria cracking for clinical myocardial ischemia-reperfusion injury protection strategy provides new theoretical basis,to realize the improvement of the overall treatment of ischemic heart disease.Materials and Methods:1.Notch1 alleviates myocardial ischemia-reperfusion injury by inhibiting mitochondrial cleavage（1）Neonatal rat cardiomyocytes were isolated from the SPF male SD rats（body weight 300±25 g）.A recombinant adenovirus expressing NICD cDNA was prepared by pAdeasy TM vector system and infected primary cardiomyocytes 50 times.（2）H/R model of cardiomyocytes was constructed by 95%N₂ and 5%CO₂ fully saturated hypoxia buffer,and 5%CO2 and 95%O₂ fully saturated reoxygenation buffer.（3）Cell viability of H/R cardiomyocytes transfected with adenovirus NICD was determined by CCK-8 method.（4）Detection of mitochondrial fusion/fission in H/R cardiomyocytes transfected with adenovirus NICD by Laser Confocal microscope（LSCM）（5）Apoptosis of H/R cardiomyocytes transfected with adenovirus NICD was detected by flow cytometry.（6）TheΔΨm of H/R cardiomyocytes transfected with adenovirus NICD was determined by flow cytometry.（7）The expression of MFN1,MFN2,NICD,OPA1,DRP1,FIS1,Cytochrome C,ANT,CYP-D,PiC and VDAC proteins in H/R cardiomyocytes transfected with adenovirus NICD was detected by Western blot.2.Notch1 alleviates myocardial ischemia-reperfusion injury by regulating the expression of MFN1 and DRP1（1）Neonatal rat cardiomyocytes were isolated from the SPF male SD rats（body weight 300±25 g）.Recombinant adenovirus expressing NICD,MFN1 and DRP1complementary DNA（cDNA）was prepared by pAdeasy TM vector system and infected primary cardiomyocytes 50 times.（2）H/R model of cardiomyocytes was constructed by 95%N₂ and 5%CO₂ fully saturated hypoxia buffer,and 5%CO₂ and 95%O₂ fully saturated reoxygenation buffer.（3）Cell viability of H/R cardiomyocytes transfected with adenovirus NICD,MFN1 and DRP1 was determined by CCK-8 method.（4）Detection of mitochondrial fusion/fission of H/R cardiomyocytes transfected with adenovirus NICD,MFN1 and DRP1 by Laser Confocal microscope（LSCM）（5）Apoptosis of H/R cardiomyocytes transfected with adenovirus NICD,MFN1and DRP1 was detected by flow cytometry.（6）TheΔΨm of H/R cardiomyocytes transfected with adenovirus NICD,MFN1and DRP1 was determined by flow cytometry.（7）Western blot was used to detect the expression of MFN1,MFN2,NICD,OPA1,DRP1,FIS1,Cytochrome C,ANT,CYP-D,PiC and VDAC proteins in H/R cardiomyocytes transfected with adenovirus NICD,MFN1 and DRP1.3.Transcription factor RBP-Jκactivates the expression of MFN1 in myocardial ischemia-reperfusion injured cells（1）Neonatal rat cardiomyocytes were isolated from the SPF male SD rats（body weight 300±25 g）.（2）H/R model of cardiomyocytes was constructed by 95%N₂ and 5%CO₂ fully saturated hypoxia buffer,and 5%CO₂ and 95%O₂ fully saturated reoxygenation buffer.（3）Interaction between promoter MFN1 and transcription factor RBP-Jκdetected by double luciferase reporter gene.（4）The interaction between promoter MFN1 and transcription factor RBP-Jκprotein-DNA was detected by CHIP method.（5）The interaction between promoter MFN1 and transcription factor RBP-Jκprotein-DNA was detected by EMSA.4.In vivo validation of the protective effect of Notch1 on myocardial ischemia-reperfusion injury by inhibiting mitochondrial cleavage（1）Neonatal rat cardiomyocytes were isolated from SD rats（1 day old）.Recombinant adenovirus expressing rat NICD,MFN1 and DRP1 complementary DNA（cDNA）was prepared by pAdeasy TM vector system and infected primary cardiomyocytes 50 times.（2）Sodium pentobarbital（70 mg/kg）was injected intraperitoneally,intubation was performed,mechanical ventilation was performed,the frequency was 60-80/min,the tidal volume was 2-3 m L/100 g,and the inhaling-breathing ratio was 1:1.2.The left fourth intercostal artery was inserted into the chest,the happy bundle was cut,6-0sliding wire was placed 2-3 mm below the intersection between the great cardiac vein and the left atrial appendage,and both ends were passed through the flexible P50 tube with a length of 0.7 cm.After 15 min of balancing,the left anterior descending coronary artery and P50 tube were ligated to form focal myocardial ischemia.After 30 min,the slip-nodule was loosened and the P50 tube was evacuated.I/R model was constructed after reperfusion for 120 min.（3）Systolic function of I/R rats was measured by left ventricular pressure.（4）Evans Blue and TTC staining were used to determine the size of myocardial infarction in I/R rats.（5）The protein expressions of MFN1,MFN2,OPA1,DRP1 and FIS1 in cardiomyocytes of I/R rats were detected by Western blot.（6）The mitochondrial structure of I/R rat cardiomyocytes was observed by scanning electron microscopy.Results:1.Notch1 alleviates myocardial ischemia-reperfusion injury by inhibiting mitochondrial cleavage（1）NICD overexpression significantly increased the viability of H/R cardiomyocytes,while NICD knockout significantly decreased the viability of H/R cardiomyocytes.（2）NICD overexpression significantly increased the number of mitochondria lengthened in H/R cardiomyocytes,while NICD knockout significantly increased the number of mitochondrial fragments in H/R cardiomyocytes.（3）NICD overexpression was significantly decreased in H/R cardiomyocytes,and significantly increased in NICD knockout H/R cardiomyocytes.（4）After NICD overexpression,the expression levels of mitochondrial fusion markers（MFN1,MFN2 and OPA1）in H/R cardiomyocytes increased,while the expression levels of mitochondrial fission markers（DRP1 and FIS1）in H/R cardiomyocytes decreased.（5）Overexpression of NICD significantly improved theΔΨm of H/R cardiomyocytes;However,down-regulation of NICD had no effect on H/R cardiomyocytesΔΨm.（6）Cytochrome C levels were significantly decreased in cardiomyocytes exposed to NICD overexpression of H/R.（7）The expression levels of ANT,CYP-D,PiC and VDAC proteins in mitochondrial permeability transition pore subunits of H/R cardiomyocytes exposed to NICD knockdown continued to decrease,indicating that the mitochondrial permeability transition pore of H/R cardiomyocytes was opened and damaged.2.Notch1 alleviates myocardial ischemia-reperfusion injury by regulating the expression of MFN1 and DRP1（1）After MFN1 deletion,the cell viability of H/R cardiomyocytes transfected with NICD overexpression was significantly impaired;However,cell viability of NICD knockdown transfected H/R cardiomyocytes increased with MFN1 overexpression.（2）Confocal mitochondrial microscopy analysis showed that MFN1 knockdown significantly reduced mitochondrial elongation in H/R cardiomyocytes transfected with NICD overexpression,while MFN1 overexpression significantly increased mitochondrial fragmentation in H/R cardiomyocytes transfected with NICD overexpression.（3）After MFN1 knockout,the apoptosis of H/R cardiomyocytes transfected with NICD overexpression was significantly increased.However,apoptosis of NICD knockdown transfected H/R cardiomyocytes decreased with MFN1 overexpression.（4）Western blot confirmed that mitochondrial fusion and cleavage markers（MFN1,MFN2,OPA1,DRP1 and FIS1）were decreased after MFN1 gene knockout in H/R cardiomyocytes transfected with NICD overexpression,but increased after MFN1overexpression in H/R cardiomyocytes transfected with NICD knockdown.（5）The activity of H/R cardiomyocytes transfected with NICD overexpression decreased with DRP1 overexpression,while the activity of H/R cardiomyocytes transfected with NICD knockdown increased with DRP1 knockdown.（6）Confocal mitochondrial microscopy analysis showed that DRP1overexpression significantly reduced extended mitochondria in H/R cardiomyocytes transfected with NICD overexpression,while DRP1 knockdown significantly increased mitochondrial fragmentation in H/R cardiomyocytes transfected with NICD knockdown.（7）In H/R cardiomyocytes transfected with NICD high expression,DRP1 high expression significantly increased the number of H/R cardiomyocytes apoptosis,while in H/R cardiomyocytes transfected with NICD knockout,DRP1 knockdown significantly reduced the number of H/R cardiomyocytes apoptosis.（8）Western blot confirmed that DRP1 overexpression was decreased in H/R cardiomyocytes transfected with high NICD expression by mitochondrial fusion and lysis markers（MFN1/2,OPA1 and FIS1）,while DRP1 was increased in H/R cardiomyocytes transfected with NICD knockdown.3.Transcription factor RBP-Jκactivates the expression of MFN1 in myocardial ischemia-reperfusion injured cells（1）RBP-Jκoverexpression significantly induced wild-type MFN1 promoter activity,but failed to induce mutant MFN1 promoter activity.（2）The direct interaction between RBP-Jκand MFN1 promoter region was evaluated by chromatin immunoprecipitation method,which indicated that MFN1promoter region was significantly enriched in RBP-Jκimmunoprecipitation component.（3）Chip-PCR assay demonstrated direct interaction between RBP-Jκand MFN1promoter region.（4）Electrophoretic migration test further confirmed the direct interaction between RBP-Jκand MFN1 promoter region.4.In vivo validation of the protective effect of Notch1 on myocardial ischemia-reperfusion injury by inhibiting mitochondrial cleavage（1）Preischemic systolic parameters were similar among all groups,while I/R（30min/45 min）inhibited left ventricular systolic function,which was characterized by LVDP,LVEDP,and the maximum rate of development and decline of left ventricular pressure（±DP/dt）,whereas NICD overexpression was significantly reduced.Preischemic-systole parameters in NICD overexpressed I/R rats were impaired by MFN1 or DRP1 overexpression,while those in NICD overexpressed RATS were conversely improved by MFN1 or DRP1 overexpression.（2）2 h after perfusion,NICD overexpression could significantly inhibit infarct size,which was similar to that of the group with NICD knockdown but MFN1overexpression or DRP1 knockdown.（3）Western blot confirmed that mitochondrial fusion markers（MFN2 and OPA1）were decreased after MFN1 knockdown,but increased after MFN1 overexpression or DRP1 knockdown.However,in the NICD overexpression group,mitochondrial fission markers（FIS1）were inversely elevated after MFN1 or DRP1 knockdown,while decreased after MFN1 overexpression.（4）Electron microscope observation of mitochondrial structure showed that many mitochondrial lesions in the HEART of I/R rats had obvious changes,including mitochondrial crest rupture and stroma swelling,indicating that mitochondrial structure was damaged.However,these impairments were attenuated in NICD overexpression and knockdown MFN1 overexpression or DRP1 knockdown.Conclusion:（1）Notch1 alleviates myocardial ischemia-reperfusion injury by inhibiting mitochondrial cleavageAdenovirus vector ad-NICD significantly increased the cell viability of H/R cardiomyocytes and the number of mitochondria lengthened.In addition,apoptotic cells were significantly reduced in NICD overexpressed H/R cardiomyocytes.In addition,Western blot showed that the expression levels of mitochondrial fusion markers（MFN1,MFN2 and OPA1）were increased and the expression levels of mitochondrial fission markers（DRP1 and FIS1）were decreased in H/R cardiomyocytes after overexpression of NICD.NICD overexpression significantly improved theΔΨm of H/R cardiomyocytes.Cytochrome C levels were significantly reduced in cardiomyocytes exposed to NICD overexpressed H/R.Exposure to NICD overexpressed H/R cardiomyocytes continuously reduced the protein expression levels of mitochondrial permeability transition pore subunits（ANT,CYP-D,PiC and VDAC）,indicating the opening and damage of mitochondrial permeability transition pore in H/R cardiomyocytes.（2）Notch1 alleviates myocardial ischemia-reperfusion injury by regulating the expression of MFN1 and DRP1Notch1,activated by RBP-Jκ-dependent MFN1 and DRP1 transcription,regulates the dynamic balance between mitochondrial fusion and fission in cardiomyocytes exposed to cardiac H/R.On the other hand,the expression of DRP1 was down-regulated and mitochondrial fission was inhibited.Mitochondrial disorders due to abnormal fusion and division lead to myocardial I/R.Our study further demonstrated that Notch1 can reduce mitochondrial cleavage,reduce the size of myocardial infarction,inhibit ventricular remodeling,and play a protective role in myocardium.This protective effect on the heart was almost reversed by MFN1 knockdown or DRP1overexpression,suggesting that the protective effect of the Notch1 signaling pathway depends on MFN1/DRP1 regulated mitochondrial fusion-fission dynamics.（3）Transcription factor RBP-Jκactivates the expression of MFN1 in myocardial ischemia-reperfusion injured cellsRBP-Jκoverexpression significantly induced wild-type MFN1 promoter activity,but failed to induce mutant MFN1 promoter activity.The promoter region of MFN1was significantly enriched in RBP-Jκimmunoprecipitation component.Direct interaction between RBP-Jκand MFN1 promoter region.（4）In vivo validation of the protective effect of Notch1 on myocardial ischemia-reperfusion injury by inhibiting mitochondrial cleavagePreischemic systolic parameters were similar among the groups,while I/R（30min/45 min）inhibited left ventricular systolic function,characterized by LVDP,LVEDP,and the maximum rate of development and decline of left ventricular pressure（±DP/dt）,whereas NICD overexpression was significantly reduced.Notably,the cardiac post-ischemic contractile parameters in NICD overexpression rats were impaired by MFN1knockdown or DRP1 overexpression,whereas those in NICD knockdown rats were conversely improved by MFN1 overexpression or DRP1 knockdown.In addition,we found that NICD overexpression significantly inhibited infarct size 2 h after perfusion,which was similar to that of the group with low NICD but MFN1 overexpression or DRP1 knockdown.Western blot confirmed that mitochondrial fusion markers（MFN2and OPA1）were decreased after MFN1 knockdown,but increased after MFN1overexpression or DRP1 knockdown.However,in the NICD overexpression group,mitochondrial fission markers（FIS1）were inversely elevated after MFN1 or DRP1knockdown,while decreased after MFN1 overexpression.Electron microscopy of mitochondrial structures revealed significant changes in many mitochondrial lesions of I/R cardiomyocytes,including mitochondrial crest rupture and stroma swelling.However,these lesions were attenuated in the NICD overexpression group and in the NICD knockdown group but MFN1 overexpression or DRP1 knockdown group.