Role Of NOTCH1in Myocardial Ischemia/Reperfusion Injury

Background:Nowadays, the incidence of coronary heart disease is increasing year by year alongwith the change of people’s living habits, therefore how to prevent and treat this diseasebecomes one hotspot in the field of cardiovascular researches. After an acute myocardialinfarction, early coronary artery reperfusion with the use of thrombolytic therapy orprimary percutaneous coronary intervention (PCI) has been widely applied as the mosteffective therapeutic approaches to reduce myocardial infarct size and improve clinicaloutcome. However, such process of reperfusing blood flow to ischemic myocardium, caninduce activation of an injurious cascade termed reperfusion injury. Undoubtedly, itparadoxically reduces the beneficial effects of myocardial reperfusion. As anevolutionarily conserved signaling system, Notch pathway has been recognized to expressin a series of organs and tissues of mammals, including hearts and blood vessels. In earlycardiogenesis, specific silencing of Notch1perturbs cardiac morphogenesis; in adult heartadaption, augmentation of Notch1activation exerts protective effects in alcoholiccardiomyopathy, myocardial infarction, and cardiac hypertrophy. In animal experiments, Notch1has been suggested to modulate various protective molecular signal pathways; incontrast, inhibition of Notch1signaling can enhance the injury in stressed hearts. Inaddition, Notch1pathway has been reported to protect hepatocytes fromischemia/reperfusion (I/R) injury. However, whether Notch1signaling exhibits protectiveeffects against the myocardial ischemia/reperfusion (MI/R) injury and its underlyingmechanism remain unclear. The present study raises important considerations regardingpossible mechanisms about Notch1signal in adult heart. Determining association betweenNotch1signal and MI/R injury and delineating the mechanism by which Notch1confersits cardioprotection constitute the focus of our study. Therefore, cardiac-specific Notch1signaling can be redeployed in the treatment of coronary heart disease.Objectives:1. To establish cardiac Notch1knockdown mice models; and to examine the effects ofNotch1on MI/R injury;2. To determine the role of oxidative/nitrative stress in the protection of Notch1againstMI/R injury;3. To investigate whether Notch1signaling participates in the cardioprotection of TNF-inhibitor in MI/R, and to elucidate the relating mechanism.Methods:1. Cardiac specific Notch1knockdown or upregulation mice models: Mice were allanesthetized with2%isoflurane in the lab, and an incision was made between the4th and5th left ribs to expose the heart.20μg of Notch1siRNA or Scrambled siRNA was dilutedin30μl of vivo-jetPEI (Invitrogen USA) and10%glucose mixture.30μl of vehiclescrambled siRNA, or Notch1siRNA solution was delivered via6separate intramyocardialinjections (5μl per injection) into the left ventricle apex and anterolateral wall using a30-gauge needle. Another groups of mice were injected with Jagged1peptide (a Notchligand,12μg diluted in30μl PBS) or PBS alone following the same procedures asdescribed above.2. Mice MI/R model: After48hours of siRNA or Jagged1injection in hearts, the incisionwas opened under anesthetic condition once again. Myocardial infarction was induced by the ligation of left anterior descending coronary artery with a slipknot for30minutes.After20minutes ischemia, mice were anesthetized with2%isoflurane again andrandomized to receive vehicle, EUK134(a ONOO-scavenger,5mg/kg, i.p.) or Etanercept(a TNF-inhibitor8mg/kg, i.p.).3. After3hours of reperfusion,(1) Myocardial apoptosis was determined by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining and caspase-3colorimetric assay kit, following the manufacturer’s instructions;(2) Western blots were performed to determine the expression of Notch1ICD, gp91phox,iNOS, eNOS/peNOS, Akt/pAkt and PTEN in cardiac tissues;(3) Myocardial superoxide content was determined by lucigenin-enhanced luminescenceand DHE staining, respectively;(4) Cardiac tissues were prepared for the quantification of NO levels. The total level ofnitrite (NO2-) and nitrate (NO3-), two major in vivo metabolites of NO, were determined torepresent overall NO production. A commercial nitrate reductase kit (JianchengBioengineering Co Ltd, Nanjing, China) was used to convert nitrate to nitrite, and GriessReagents were used to convert nitrite to a deep purple azochromophore;(5) Nitrotyrosine is recognized as a footprint of in vivo peroxynitrite formation, thus it wasused as an index for nitrative stress in reperfused heart tissues. The assessment ofnitrotyrosine content was realized via chemiluminescent and immunohistological analysis,respectively;(6) Mitochondrial morphology (cristae and membrane) were examined by transmissionelectron microscopy (TEM);(7) Mitochondrial function was determined by ATP content and membrane potential,which were examined by ATP kit and JC-1kit respectively.4. After24hours of reperfusion,(1) Mice were re-anesthetized with isoflurane and left ventricular ejection fraction (LVEF)and left ventricular fractioning shortening (LVFS) were obtained by transthoracicechocardiography. (2) After functional determination, the ligature around the coronary artery was retied again,and then myocardial infarct size was evaluated by Evans blue/the2,3,5-triphenyltetrazolium chloride (TTC) double staining method.5. After2weeks, cardiac function was determined again for those remained mice as abovementioned. Paraformaldehyde-fixed cardiac tissues were cut into sections of4~5μm thickand stained with Masson’s trichrome (MT).Results:1. The expression level of Notch1ICD (Notch1intracellular domain), activated form ofNotch1, was measured to determine the efficiency of Notch1siRNA or Jagged1. Whencompared to normal MI/R, Notch1signaling was successfully downregulated by45.23%2days after Notch1siRNA injection, as evidenced by markedly decreased Notch1ICDexpression; in addition, Notch1signaling was also significantly increased by77.07%2days after Jagged1injection, as evidenced by markedly increased Notch1ICD expression.2. When compared to normal MI/R, after3hours of reperfusion: TUNEL staining andCaspase-3activity assay revealed remarkable increase in myocardial apoptosis in cardiacspecific Notch1knockdown mice; Notch1inhibition enhanced the disruption ofmitochondrial morphology, lessened ATP content and decreased mitochondrial potential,indicative of aggravated sub-cellular organelle; downregulation of Notch1increased theexpression of iNOS, gp91phoxand PTEN, enhanced the production of NO and superoxide,as well as their cytotoxic reaction product peroxynitrite, indicative of increasedoxidative/nitrative stress level; in contrast, Notch1blockade also reduced phosphorylationof eNOS and Akt, indicative of inhibition of protective signaling. After24hours ofreperfusion, knockdown of cardiac specific Notch1further increased myocardial infarctsize and enhanced functional impairment post-MI/R. After2weeks of reperfusion,knockdown of cardiac specific Notch1further enhanced MI/R-induced increase of cardiacfibrosis and depression of cardiac function.3.10minutes before reperfusion, Notch1knockdown mice were treated with EUK134viaintraperitoneal injection. As a result, EUK134markedly attenuated MI/R injury, asevidenced by attenuation of mitochondrial impairment, reduced Caspase-3activity and infarct size, and improvement of cardiac function. Moreover, EUK134significantlyreduced myocardial nitrotyrosine content in Notch1knockdown mice subjected to MI/R,suggesting that enhancing peroxynitrite decomposition could effectively reduceperoxynitrite-induced protein nitration in cardiac tissues.4. To deeply investigate Notch1signaling pathway in our study, an additional experimentwas performed by using intramyocardial injection of Jagged1in normal mice48hoursbefore MI/R. As expected, Jagged1successfully activated Notch1signal in I/Rmyocardium. Moreover, Jagged1injection markedly attenuated MI/R injury, asdetermined by attenuation of mitochondrial impairment, decreased Caspase-3activity andinfarct size, and improvement of myocaridal function. Most importantly, Jagged1administration significantly increased eNOS phosphorylation at serine1177, whereasdecreased iNOS expression, superoxide generation, NO production and nitrotyrosinecontent. These results suggest that Notch1activation with its ligand can increasemyocardial resistance to MI/R injury.5. Mice were randomly received vehicle or Etanercept administration via intraperitonealinjection10minutes before reperfusion. Etanercept successfully attenuated myocardialinjury in mice subjected to I/R, evidenced by attenuation of mitochondrial impairment,decreased Caspase-3activity and infarct size, and improvement of cardiac function. Inaddition, Etanercept decreased the expression of iNOS and gp91phox, lessened theproduction of NO and superoxide, as well as their cytotoxic reaction product peroxynitrite.Whereas, downregulation of Notch1significantly reversed the attenuation of MI/R injuryinduced by Etanercept and increased oxidative/nitrative stress level in cardiac tissues.These data indicate that MI/R injury can be attenuated by TNF-inhibitor partly viaNotch1signaling mediated suppression of oxidative/nitrative stress.Conclusions:1. Notch1signaling pathway responds to MI/R injury; endogenous Notch1signaling iscritical to promote cardiomyocyte survival, inhibit cardiac fibrosis, and sustain cardiacfunction;2. Notch1signaling protects against MI/R injury partly though PTEN/Akt mediated anti-oxidative/nitrative effects;3. TNF-inhibitor can activate Notch1pathway in MI/R; and Notch1signalingparticipates in the cardioprotection of TNF-inhibitor in MI/R partly viaanti-oxidative/nitrative effects.