Study On Myocardial Ischemia/Reperfusion Injury And Regulation Of Calcineurin Signaling Pathway

After an acute ischemic heart disease, early and successful myocardial reperfusion is an effictive strategy for obtaining thoroughly coronary blood flow, saving the dying myocardiocyte and reducing the size of a myocardial infarct. Because of the uncertainty relates to the ischemic period and degree, myocardial ischemia/reperfusion injury caused by the restoration of coroary blood flow after an ischemic episode. This form of myocardial injury can induce cardiomyocyte apoptosis and necrosis, increase infarct size, remodel cardiac ventricle, then the ultmate incidence is cardiac failure. To observe H₂O₂-induced myocardial H9c2 cell apoptosis and pretective effect of inhibitor of calcineurin (CaN), a cell apoptotic model from rat myocardiocytes cell line H9c2 induced by hydrogen dioxide (H₂O₂) and a rat model of ischemia/reperfusion are established. These provide theoretical basis for study on CaN induced myocardiocyte apoptosis caused by ischemia/reperfusion indury and related mechanism.1.Toxicity of H₂O₂ on H9c2 cells1.1 The survival rate of H9c2 cell detected by MTT MTT assay was performed to detect the toxicity after 1, 3, 6, 12 and 24 h exposure to H₂O₂ (0, 50, 100, 200 and 400μmol/L). It was showed the survival rate decreased with the increase of dosage and duration of exposure to H₂O₂. After 6 h exposure to 200μmol/L H₂O₂,half of the H9c2 cells died. The change of survival rate was over 60%, with 24 h after exposure 400μmol/L H₂O₂. There was significant difference in survival rate between H₂O₂ group and control group(NC) (P < 0.05). The results suggest that cardiomyocytes are sensitive to H₂O₂-induced dosage.1.2 DNA damage of H9c2 cellsSingle cell gel electrophoresis (SCGE) assay was used to examine DNA damage of H9c2 cells 6 h after exposure to H₂O₂ (0, 50, 100, 200 and 400μmol/L). When exposed to 50, 100, 200 and 400μmol/L H₂O₂ for 6 h, there were significant difference from control group. The results suggest H₂O₂ could cause DNA damage of myocardiocytes in dose dependant way.1.3 Apoptosis detected by AO/EB double fluorescent staining assay There were distinctive changes on cell apoptosis observed under fluorescence microscope 1, 3, 6 and 12 h after exposure to H₂O₂ (0, 50, 100, 200 and 400μmol/L) by AO/EB double staining. The H9c2 cells occur obviously morphological changes in H₂O₂ -exposed groups, and the apoptotic rate was a significant increase compared with control group (P < 0.05), the highest group at 6 h after exposure to 100μmol/L H₂O₂. Whthin a prolong time exposure to it, the change of apoptotic rate is not obvious.1.4 Apoptosis detected by FCM with Annexinâ…¤/PI double fluorescent staining Cell apoptosis was observed by FCM 1, 6 and 24 h after exposure to H₂O₂ (0, 50, 100, 200 and 400μmol/L). The apoptotic rates were significantly increased in 100, 200 and 400μmol/L H₂O₂ at 6 and 24 h compared with control group(P < 0.05), the highest group at 24 h after exposure to 400μmol/L H₂O₂. The results suggest H₂O₂ could induce the H9c2 cell apoptosis in dose dependant and time dependant way.1.5 Membrane potential detected by FCM with Rhodamine 123 fluorescent prodeMitochondria membrane potential (Δψmt) was observed by FCM 1, 6 and 24 h after exposure to H₂O₂ (0, 50, 100, 200 and 400μmol/L).Δψmt showed the significant increase in H₂O₂ -exposed groups at 1 h compared with control group (P < 0.05). Whthin a prolong time exposure to it, the change ofΔψmt is observed. Compared with control group,Δψmt showed the significant increase in 50 and 100μmol/L H₂O₂ at 6 h, and the significant decrease in 200 and 400μmol/L H₂O₂ at 24h (P < 0.05). The results suggest H₂O₂ could damage mitochondria, decreaseΔψmt and induce the apoptosis in myocardial H9c2 cell.2. Effect of inhibitor of calcineurin on myocardial H9c2 cell apoptosis induced by H₂O₂The myocardial cell apoptosis models of rats were established, that is, myocardial H9c2 cells was exposed to 100μmol/L H₂O₂ at 6 h. FK506 were administerted to the model group at low dose (0.15μmol/L) and high dose (0.60μmol/L) levels, and the apoptotic and necrotic rates were detected by FCM with Annexinâ…¤/PI double fluorescent staining. Compared with 100μmol/L H₂O₂-exposed group at 1 h, the H₂O₂-induced apoptotic rate was significantly decreased in FK506 treated group (P < 0.05). The inhibitor of CaN shows protective effect on myocardial H9c2 cell apoptosis induced by H₂O₂, with dose-undependant manner.3. The protective effect on myocardial ischemia/reperfusion injury of inhibitor of CaN in rats3.1 A rat heart model of ischemia/reperfusion injury established Weigh and anesthetized the rat , then fix it on the operation table and removed the fur from the neck and the left chest. The trachea was intubated and arterial blood pressure was continuously monitored via a saline-filled catheter inserted into the common carotid artery. The chest was opened and the pericardium was incised. The heart was gently exteriorized by applying pressure on the right side of the chest, and a ligature wa placed around the left anterior descending coronary (LAD) occlusion 30 min and reperfusion 120 min. Then a rat model of myocardial ischemia/reperfusion injury was been established. Wistar rats were randomly divided into four groups: I/R group, FK506-I/R group (FK506 was intravenouosly injected 15 min before ischemia), Sham group (a ligature was placed around LAD and no occluded), FK506-Sham group (FK506 was intravenously injected , a ligature was placed around LAD and no occluded).3.2 Monitoring mean blood pressure, heart rate and electrocardiogram Mean blood pressure (MBP) was continuously monitored via an pressure transducer connected to a physiograph. Electrocardiogram and heart rate (HR) are measured by Leadâ…¡using an electrocardiogram/rate coupler, both being analyzed by the BL-410 BioLab System. Four period of MBP and HR were selected (baseline, before occlusion, end-ischemia, end-reperfusion). Evaluation of arrhythmias were based on the description of the Lambeth Conventions. Total number of episodes for premature ventricular contraction (PVC), total duration for ventricular tachycardia (VT) and ventricular fibrillation (VF) were evaluated. The severity of arrythmias was quantified by a scoring system. During the ischemia period, MBP was a significant decrease in I/R group compared with in FK506-I/R group (P < 0.05). But during the reperfusion period, these two groups were no significant difference (P > 0.05). During the four period, the changes of HR were not obvious. Administration of FK506 before I/R significantly reduced the occurrence of PVC, VT, VF and duration of cardiac arrhythmia, and reduced the arrhythmia score (P > 0.05). The results suggest FK506 shows antiarrhythmic and cardioprotective effects in rat model of myocardial ischemia/reperfusion injury.3.3 Area at risk myocardial infarct size determinationThe area at risk (ischemic/reperfused region) and the infarct size were measured using Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) double staining method. The images of heart slices were captured by a camera and area of infarcted myocardium was digitally measured using Image-Pro plus. Area-at-risk (AAR)/left-ventricular area (LV)×100%, infarct zone (IS)/LV×100%, IS/AAR×100% were calculated. Compared with I/R group, IS/AAR in FK506-I/R group was singnificant decrease (P < 0.05). The result indicated FK506 signifivantly reduced myocardial ischemia/reperfusion injury.3.4 Assay of myocardial enzyme leakageAt the end of reperfusion, blood samples were collected to measure the myocardial enzyme leakage, including creatinine kinase (CK) and lactate dehydrogenase (LDH). CK and LDH kits were applied to examine their activities. It was showed the activities of CK and LDH were siginificant decrease in FK506-I/R group compared with I/R group (P < 0.05).3.5 Lipid peroxidation of plasma examinationSOD kit was applied to examine activities of SOD in rats plasma. It was showed the activities of SOD were significant decrease in both FK506-I/R and I/R groups, compared with Sham group (P < 0.05). There were no significant changes from FK506-I/R to I/R group(P > 0.05). TBA colorimetric method was applied to examine MDA contents. It was showed the MDA contents were significant increase in both FK506-I/R and I/R groups, compared with Sham group (P < 0.05). There were no significant changes from FK506-I/R to I/R group(P > 0.05).3.6 Changes of CaN enzyme activitesCaN kit was applied to examine activites of CaN in myocardium tissues. The activities of CaN were significant decrease in FK506-I/R group compared with I/R group(P < 0.05).4. CaN induced myocardiocyte apoptosis caused by ischemia/reperfusion indury and related mechanism.4.1 Changes of expression of Bcl-2, Bax, and Caspase– 3 Immunocytochemical method was used to detect expression of Bax, Bcl-2 and Caspase-3. It could be observed under optical microscope that nucleus of positive expressed cells was dyed dark blue and cytoplasm or cellular membrane was dyed brown. Expression of Bcl-2 significantly increased in FK506-I/R group, and expression of Bax significantly increased in I/R group compared with Sham group(P < 0.05). The value of Bcl-2/Bax were a significant difference between FK506-I/R and I/R group(P < 0.05). Expression of Caspase-3 was a significant increase in FK506-I/R and I/R group compared Sham group (P < 0.05). The result suggests FK506 could depress myocardiocyte apoptosis.4.2 Changes of expression of CaNWestern blot was applied to detect CaN activity, and the characteristic 62 kDa fragment of CaN was observed in four groups. The expression of CaN enhanced and got to the highest in I/R group, the lowest in FK506-Sham group.Generally, H₂O₂ could cause oxidative injury and DNA damage. It could also cause mitochondrial damage, then decrease mitochondrial mambrane potential. So H₂O₂ could induce the apoptosis on myocardial H9c2 cells. The inhibitor of CaN, FK506, could suppress H₂O₂-induced apoptosis on the H9c2 cells, and shows cardioprotective effect . FK506 could reduce the size of infarct zone on a rat model of myocardial ischemia/reperfusion injury, and increase the expression of Bcl-2 protein. So the inhibitor of CaN could suppress myocardiocyte apoptosis, the mechanism is related to CaN production and induced apoptosis by mitochondria signal transduction.