The Effect And Mechanism Of Trimetazidine On Myocardial Ischemia Reperfusion Injury

Myocardial ischemic injury results from severe impairment of the coronary blood supply usually produced by thrombosis or other acute alterations of coronary atherosclerotic plaques.It is the main factor leading to myocardial infarction,ischemic stroke and organ transplantation injury.Intense and sustained investigation has produced considerable insight into the pathobiology of myocardial ischemic injury.Rapid and effective recovery of blood supply to ischemic myocardium is still the most effective method to treat myocardial ischemia.Cutaneous coronary angioplasty,arterial bypass and thrombolytic therapy are often used to reconstruct the blood supply of ischemic myocardium.However,reperfusion itself can cause new and additional damage to the ischemic myocardium,known as reperfusion injury,which can greatly affect the therapeutic effect.Therefore,it is of great clinical significance to clarify ischemia-reperfusion injury.Mitochondria are the metabolic and energy centers of cells,especially cardiomyocytes,and the main sites for ATP production in cells.They contain many other metabolic networks and play an important role in regulating apoptosis and necrosis of cells.In addition,mitochondria are also one of the main sources of reactive oxygen species（ROS）.The production of a large amount of ROS will damage mitochondria,resulting in metabolic disorders,energy disorders,apoptosis or necrosis of cells,which is related to the occurrence and development of many diseases.The involvement of ROS in ischemia-reperfusion injury has attracted the attention of many researchers.The large accumulation and release of mitochondrial ROS during reperfusion is an important cause of reperfusion injury,which triggers many downstream pathological processes in the process of ischemia-reperfusion injury.A better understanding of the mechanism of ROS production and its relationship with ischemia reperfusion injury is crucial to understanding how oxidative stress promotes ischemia reperfusion injury.Traditional drugs for stable angina include symptomatic therapies that control heart rate,such as dioxide-beta blockers,non-dihydropyridine calcium channel antagonists,and vasodilator ivarabendine,which selectively inhibits the sinoatrial node If channel.Trimetazidine plays an anti-ischemic role by regulating cardiac metabolism without changing hemodynamic function,which is a good supplement to traditional treatment of angina pectoris.It also improves inflammatory response and endothelial function.Trimetazidine can also be used to treat patients with coronary artery reconstruction or left ventricular dysfunction and diabetes.Trimetazidine is an anti-ischemic drug widely used in the treatment of coronary heart disease.By inhibiting the long chain 3-ketoacyl coenzyme A mercaptan enzyme in mitochondria,the uptake and oxidation of myocardial fatty acids were inhibited,thereby activating glucose oxidation and changing the metabolism of mitochondrial energy substrates.Unlike traditional drugs,trimetazidine has no effect on coronary flow,systolic force,blood pressure or heart rate.There was no significant negative muscle strength or vasodilation during rest or exercise.Thus,trimetazidine may be used in combination with conventional drugs to treat coronary artery disease as a complementary or alternative therapy when conventional drugs are not tolerated.However,there are few reports on the protective effect of trimetazidine in ischemia reperfusion injury,and the specific mechanism of its action is still unclear,which needs to be further studied and elucidated.SIRT3,one of the important members of sittuins family,is located in mitochondria and is a soluble mitochondrial NAD⁺-dependent deacetylase,which is highly expressed in the tissues rich in mitochondria and can participate in oxidative stress,fatty acid metabolism,mitochondrial energy metabolism and cell death.In normal myocardium,mitochondrial oxidative phosphorylation provides about 90%of the ATP needed for cardiac activity,and the main energy source is the oxidation of fatty acids.SIRT3 can regulate the activity of enzymes involved in oxidative phosphorylation in mitochondria by deacetylation.However,the role of SIRT3 in the improvement of myocardial ischemia reperfusion injury with trimetazidine remains unclear and needs to be clarified.Objective1.To demonstrate the protective effect of Trimetazidine（TMZ）on rat isolated heart ischemia reperfusion injury,and search for the optimal drug concentration.2.To explore the mechanism of the protective effect of trimetazidine in reducing oxidative stress on ischemia reperfusion injury in isolated rat heart,and to explore whether the SIRT3-SOD signalling involved.Part 1:Trimetazidine alleviated ischemia reperfusion injury in isolated rat heart MethodsMale healthy SD rats weighing between 220 g and 250 g were purchased from the experimental animal center of the Air Force Medical University,and were fed adaptably in the laboratory animal room for 3 days before the experiment.The rats were rapidly intraperitoneally injected with chloral hydrate（100 mg/ml）and heparin（500 U/kg）.After waiting for anesthesia,the rats underwent thoracotomy.The hearts were rapidly exposed and excised,and immediately fixed to the aortic cannula of the Langendorff perfusion system.After the normal perfusion of isolated heart began,the experiment was randomly divided into the following 5 groups:1.Control group:The isolated hearts were received normal perfusion solution for 160minutes;2.Ischemia reperfusion group（IR）:After the isolated hearts stabilization with perfusion solution,the hearts were subjected to 40 minutes of global ischemia followed by120 minutes of reperfusion.3.5μmol/L trimetazidine treatment with ischemia reperfusion group（TMZ-5+IR）:After the isolated hearts stabilization with perfusion solution,5μmol/L trimetazidine perfusion solution was administrated for 1 minute,the hearts were subjected to 40 minutes of global ischemia,during the first 5 minutes of reperfusion,5μmol/L trimetazidine perfusion solution was administrated,after then the hearts were perfused with normal perfusion solution;4.10μmol/L trimetazidine treatment with ischemia reperfusion group（TMZ-10+IR）:After the isolated hearts stabilization with perfusion solution,10μmol/L trimetazidine perfusion solution was administrated for 1 minute,the hearts were subjected to 40 minutes of global ischemia,during the first 5 minutes of reperfusion,10μmol/L trimetazidine perfusion solution was administrated,after then the hearts were perfused with normal perfusion solution;5.20μmol/L trimetazidine treatment with ischemia reperfusion group（TMZ-20+IR）:After the isolated hearts stabilization with perfusion solution,20μmol/L trimetazidine perfusion solution was administrated for 1 minute,the hearts were subjected to 40 minutes of global ischemia,during the first 5 minutes of reperfusion,20μmol/L trimetazidine perfusion solution was administrated,after then the hearts were perfused with normal perfusion solution.The values of left ventricular pressure was recorded by Labchart software,and left ventricular development pressure（LVDP）and left ventricular end-diastolic pressure（LVEDP）was used to evaluate the cardiac function.The lactate dehydrogenase（LDH）content was recovered 10 minutes before the begin of reperfusion.At the end of reperfusion,the myocardial infarction area was determined by TTC staining,and the caspase-3 activity in the myocardial tissue of each group was detected 30 minutes after reperfusion.After 10 minutes of reperfusion,the left ventricular myocardial tissue was splitted for western blotting.The protein levels of cytochrome c and cleaved caspase-3were detected by western blotting.These results can be used as indicators to evaluate the degree of myocardial cell necrosis and apoptosis in each group.Results1.Compared with the Control group,the IR group showed a significant decrease in cardiac function,as manifested by a decrease in LVDP and an increase in LVEDP;Compared with the IR group,IR treatment with 5μmol/L TMZ group showed an increase in LVDP and a decrease in LVEDP,but there was no statistical difference.Both IR treatment with 10μmol/L TMZ group and IR treatment with 20μmol/L TMZ group could significantly increase LVDP and significantly reduce LVEDP,but there is no statistical difference between these two groups.2.Compared with the Control group,the myocardial infarction area of the IR group was significantly increased,the content of LDH in the coronary outflow,the caspase-3activity,and the protein expression of cytochrome c and cleaved caspase-3 were also significantly increased.Compared with the IR group,IR treatment with 5μmol/L TMZ group showed a tendency to reduce myocardial infarction area,LDH content in coronary outflow,caspase-3 activity and cytochrome c and cleaved caspase-3 protein expression,but there was no statistical difference.Both IR treatment with 10μmol/L TMZ group and IR treatment with 20μmol/L TMZ group could significantly reduce the myocardial infarction area,LDH content in coronary outflow,caspase-3 activity and cytochrome c and cleaved caspase-3 protein expression,but there is no statistical difference between these two groups.Part 2:Trimetazidine alleviated ischemia reperfusion injury through reducing oxidative stress by activating SIRT3-SOD in isolated rat heartMethodsMale healthy SD rats weighing between 220 g and 250 g were purchased from the experimental animal center of the Air Force Medical University,and were fed adaptably in the laboratory animal room for 3 days before the experiment.The rats were rapidly intraperitoneally injected with chloral hydrate（100 mg/ml）and heparin（500 U/kg）.After waiting for anesthesia,the rats underwent thoracotomy.The hearts were rapidly exposed and excised,and immediately fixed to the aortic cannula of the Langendorff perfusion system.After the normal perfusion of isolated heart began,the experiment was randomly divided into the following 4 groups:1.Control group:The isolated hearts were received normal perfusion solution for 160minutes;2.Ischemia reperfusion group（IR）:After the isolated hearts stabilization with perfusion solution,the hearts were subjected to 40 minutes of global ischemia followed by120 minutes of reperfusion.3.10μmol/L trimetazidine treatment with ischemia reperfusion group（TMZ+IR）:After the isolated hearts stabilization with perfusion solution,10μmol/L trimetazidine perfusion solution was administrated for 1 minute,the hearts were subjected to 40 minutes of global ischemia,during the first 5 minutes of reperfusion,10μmol/L trimetazidine perfusion solution was administrated,after then the hearts were perfused with normal perfusion solution;4.10μmol/L 3-TYP and 10μmol/L trimetazidine treatment with ischemia reperfusion group（3-TYP+TMZ+IR）:After the isolated hearts stabilization with perfusion solution,10μmol/L 3-TYP perfusion solution was administrated for 1 minute,the hearts were subjected to 40 minutes of global ischemia,during the first 5 minutes of reperfusion,10μmol/L 3-TYP perfusion solution was administrated,then the hearts were perfused with was administrated 10μmol/L trimetazidine perfusion solution.The values of left ventricular pressure was recorded by Labchart software,and left ventricular development pressure（LVDP）and left ventricular end-diastolic pressure（LVEDP）was used to evaluate the cardiac function.The lactate dehydrogenase（LDH）content was recovered 10 minutes before the begin of reperfusion.At the end of reperfusion,TTC staining was used to determine the myocardial infarction area and TUNEL staining was used to examine the apoptotic index.The caspase-3 activity and ROS production in the myocardial tissue of each group were detected 30 minutes after reperfusion.After 10 minutes of reperfusion,the left ventricular myocardial tissue was splitted for western blotting.The protein levels of cytochrome c,cleaved caspase-3,SIRT3,Ac-SOD,SOD,gp91^phox were detected by western blotting.These results can be used as indicators to evaluate the degree of myocardial cell necrosis and apoptosis in each group.Results1.Compared to the Control group,the IR group showed a significant decrease in cardiac function,manifested by a decrease in LVDP and an increase in LVEDP;Compared to the IR group,the IR group treated with 10μmol/L TMZ significantly increased the LVDP and significantly reduced the LVEDP.Compared to the IR treatment with TMZ group,SIRT3 specific inhibitor 3-TYP combined with TMZ treatment IR group could block the effect of TMZ on improving IR-induced cardiac function,which is manifested as decreased LVDP and increased LVEDP.2.Compared to the Control group,the IR group showed significantly increased in myocardial infarction area,LDH content in the coronary outflow,the caspase-3 activity,the protein expression of cytochrome c and cleaved caspase-3,and theapoptotic index.Compared to the IR group,IR treatment with 10μmol/L TMZ significantly reduced the myocardial infarction area,LDH content in coronary outflow,caspase-3 activity,cytochrome c and cleaved caspase-3 protein expression and apoptotic index.However,compared to the TMZ+IR group,the 3-TYP+TMZ+IR group could block the protective effect of TMZ,as showed increased in myocardial infarction area,LDH content in coronary outflow,caspase-3 activity,cytochrome c and cleaved caspase-3 protein expression,and apoptotic index.3.Compared to the Control group,it was found that oxidative stress was enhanced in the IR group,as manifested by increased ROS production and MDA content and decreased SOD activity.Compared to the IR group,IR treatment with 10 mol/L TMZ significantly reduced ROS production and MDA content,and increased SOD activity.Compared to the TMZ+IR group,the 3-TYP+TMZ+IR group could block the anti-oxidative stress effect of TMZ,as evidenced the increase of ROS production and MDA content,and the decrease of SOD activity.4.Compared to the Control group,SIRT3 protein level was decreased in the IR group,while the protein expressions of Ac-SOD2 and gp91^phox was increased.Compared to the IR group,the TMZ+IR group significantly increased the protein expression of SIRT3 and decreased the protein expressions of Ac-SOD2 and gp91^phox.Compared to the TMZ+IR group,the 3-TYP+TMZ+IR group could block the activation of SIRT3-SOD signalling by TMZ,as showed decreased protein expression of SIRT3 and increased expressions of Ac-SOD2 and gp91^phox.Conclusion1.First,this study confirmed the protective effect of trimetazidine in isolated rat hearts ischemia reperfusion injury,and evaluated the optimal concentration of trimetazidine from the perspectives of cardiac function,apoptosis and necrosis.2.Based on the first part of the study,this study found that trimetazidine could reduce ROS production and oxidative stress by activating mitochondrial SIRT3-SOD signalling,thus playing an important protective against myocardial ischemia reperfusion injury.In conclusion,trimetazidine can reduce oxidative stress and improve myocardial ischemia reperfusion injury through SIRT3-SOD signalling,suggesting that trimetazidine can be used as an alternative drug for clinical treatment of reperfusion injury.