The Effect Of Telmisartan On Myocardial Ischemia/Reperfusion Injury In A Rabbit Model

Acute myocardial infarction (AMI) is directly caused by coronary arterial thrombosis on the base of atherosclerotic plaque ruptures or fissures. Thus primary percutaneous coronary intervention (PCI) and thrombolysis therapy have become the best reperfusion therapy for acute myocardial infarction with the aim of the restoration of the coronary artery blood flow and the myocardial tissue reperfusion as soon as possible. However, the ischemia/reperfusion (I/R) has also been associated with severe myocardial injury.Moreover, the no-reflow or slow-flow (≤TIMIⅡgrade,not TIMIⅢgrade as normal) phenomenon has been also found in coronary artery of AMI patients who undergo reperfusion therapy, the myocardial tissue reperfusion is not effective,although the coronary artery is not obstructive anymore. It is considered that the no-reflow phenomenon is associated with microcirculation disturbance. The principal mechanism of microcirculation disturbance include: damage and engorgement in vascular endothelial cells , angiospasm in micrangium, neutrophils adherence and aggregation resulting in obstruction of vas capillare lumens. Besides,the microcirculation disturbance would also induce myocardial ischemia/reperfusion (I/R) injury, which would lead to thoroughly myocardiolysis,infarct expansion and the aggravation of functional disturbance in left ventricle.Nowadays,it has been demonstrated that the renin-angiotensin system (RAS) was responsible for myocardial I/R injury,angiotensin II (Ang II) is the primary active peptide hormone of this system. Angiotensin II generates the biological effect by the activation of angiotensinⅡtype 1 receptors (AT1R) and angiotensinⅡtype 2 receptors (AT2R). More and more evidence has showed that interaction existed between AT1R and AT2R. Furthermore,AT2R has the opposite effect to AT1R. Nuclear factor-κB (NF-κB) plays an important role in myocardial I/R injury. The IκB kinaseβ(IKK-β) is the key enzyme of the NF-κB activation.All the ARB used in clinic blocked the activation of AT1R selectively, but not blocked the effect of AT2R. It has been demonstrated that ARB is also an antiinflammatory which could broaden coronary artery and have protection effect on endotheliocyte. Telmisartan has special pharmacological characteristics which are superior to other ARB, including longer half life, higher trough-to-peak ratio, higher smoothness index and not excrete through renal.The cardiac pharmacological preconditioning stimulate and simulate the endogenous ptotectants to play the protection role for heart,which has more advantages including simple process, tiny damage and widely use in clinic.Therefore, the purpose of our study was to investigate the effect of telmisartan preconditioning on myocardial ischemia/reperfusion injury in a rabbit model. The study was divided into three parts. Part One was the effect of telmisartan on angiotensin receptor and IκB kinaseβof myocardial ischemia/reperfusion in a rabbit model. Part Two was the effect of telmisartan on microcirculation disturbance of myocardial ischemia/reperfusion in a rabbit model and Part Three was the effect of telmisartan on myocardial ischemia/reperfusion injury in a rabbit model. Since it was found that telmisartan had the protection effect on myocardial ischemia/reperfusion injury in a rabbit model , our study will investigate the effect of telmisartan preconditioning on myocardial AT1R, AT2R and IKK-βfirstly,then investigate the effect of telmisartan preconditioning on microcirculation disturbance,thus further investigate the effect of telmisartan on myocardial ischemia/reperfusion injury.Part One: The effect of telmisartan on angiotensin receptor and IκB kinaseβof myocardial ischemia/reperfusion in a rabbit modelObjective To investigate the effect of telmisartan preconditioning on AT1R, AT2R and IKK-βof myocardial ischemia/reperfusion in a rabbit model.Methods Thirty male New Zealand white rabbits were randomly divided into three groups, the sham operation group, the myocardial ischemia/reperfusion model group and the telmisartan treat group, ten rabbits in each group. The sham operation group and the myocardial ischemia/reperfusion model group was gavaged with physiologic saline 5ml at fixed time every day for two weeks, the treated group was gavaged with telmisartan 5mg/kg, which dissolve in physiologic saline 5ml, at fixed time every day for two weeks until the trial end. The anterior descending branch of left coronary artery (LAD) was ligated in model group and treated group but not in sham group. Rabbits in model group and treated group were subjected to 1 hour of LAD occlusion, and 6 hours of reperfusion. In the sham group, the heart was exposed after the chest opened for 7 hours without LAD ligated. The renin activity (RA) and AngⅡconcentration measured by radioimmunoassay was recorded in the plasma (carotid artery) at baseline, 1h after occlusion and 30min, 3h and 6h after reperfusion. At the endpoint of experiment (after 7 hours) the rabbits were killed, the cardiac muscle samples were removed from reperufsion zone and made into homogenate for the detection of RA and AngⅡconcentration by radioimmunoassay. Also, the cardiac muscle samples removed from reperfusion zone were processed with routine histological methods and prepared for the detection of the protein expression of AT1R, AT2R and IKK-βby immunohistochemical method.Result During the course of model building, two rabbits were died in anesthetic accident and operation accident in sham group, two rabbits were died in operation accident and ventricular fibrillation in model group, one rabbit was excluded because the data was incomplete in model group, two rabbits were died in operation accident and ventricular fibrillation in treated group, all these rabbits were excluded in the study. Eventually twenty three male New Zealand white rabbits were included in our study, eight rabbits in sham group, seven rabbits in model group and eight rabbits in treated group. In plasma, RA and AngⅡcontents were significantly increased in model group and treated group at 1h after occlusion and 30min, 3h, 6h after reperfusion compared with sham group (P < 0.01), whereas there was no significant difference between model group and treated group at 1h after occlusion and 30min, 3h, 6h after reperfusion (P < 0.05). In myocardium, RA and AngⅡcontents were significantly increased in model group and treated group at 6h after reperfusion compared with sham group (P < 0.01). Whereas there was no significant difference between model group and treated group at 6h after reperfusion (P > 0.05). Compared with sham group, the expression of AT2R protein decreased in model group, whereas enhanced in treated group (P < 0.01). Compared with sham group, the expression of IKK-βprotein increased in treated group, while it reached the highest level in model group (P < 0.01). The expression of AT1R protein had not significant difference among the three groups (P > 0. 05).Conclusion The RAS is activated during the course of myocardial ischemia/reperfusion. RA and AngⅡcontents are significantly increased in plasma and myocardium. The increased AngⅡaffects AT1R activation, increases the activation of IKK-βprotein and down-regulats the expression of AT2R protein. The telmisartan preconditioning on myocardial ischemia/reperfusion in the rabbit model depresses the activation of IKK-βprotein and upregulats the expression of AT2R protein.Part Two: The effect of telmisartan on microcirculation disturbance of myocardial ischemia/reperfusion in a rabbit model.Objective To investigate the effect of telmisartan preconditioning on microcirculation disturbance of myocardial ischemia/reperfusion in a rabbit model.Methods Thirty male New Zealand white rabbits were randomly divided into three groups. The animal model grouping, the surgical procedure and the sample size were same to those in the Part One. The animals were killed at the end of experiment. The myocardium tissue samples were removed from reperfusion zone. After disposal with routine histological methods and hematoxylin-eosin (HE) staining, the structure of vas capillare was examined and quantity of neutrophils was counted by light microscopy. The vascular endothelial cell was displayed by labelled with CD34 immunohistochemically, their quantitative analysis about the cross section areas of vas capillare was conducted by pathological imaging analyzing system. The ultramicrostructure of capillary was observed by transmission electron microscope.Result It was showed by light microscopy that in sham group, the structure of arterioles and veinules was integrated and there was no neutrophil in vas capillare lumen. In model group, a great quantity neutrophils, platelets and vascular endothelial cells adhered and aggregated in the arterioles and veinules so that vas capillare lumen was obstructed. In treated group, a small quantity neutrophils, platelets and vascular endothelial cells adhered in the arterioles and veinules. Compared with sham group, neutrophils count was significantly increased in model group (P < 0.01). Compared with model group, neutrophils count was significantly decreased in treated group (P < 0.01). Compared with sham group, the section areas of arterioles, veinules and blood capillaries were significantly decreased in model group (P < 0.01). Compared with model group, the section areas of arterioles, veinules and blood capillaries were significantly increased in treated group (P < 0.05). It was show in the transmission electron microscope that vascular endothelial cells were swelling and capillary lumen was obstructed by neutrophils in the model group. The engorgement of vascular endothelial cells was relieved in treated group. It was found that neutrophils decreased in capillary of treated group. It was not found capillary lumen was obstructed by neutrophils in treated group. The structure of vascular endothelial cells was normal in sham group.Conclusion Telmisartan preconditioning reduces the aggregation and adherence of neutrophils, expands the vas capillare, relieves the vascular endothelial cells from swelling, thus inhibites the microcirculation disturbance of myocardial ischemia/reperfusion in the rabbit model.Part Three: The effect of telmisartan on myocardial ischemia/reperfusion injury in a rabbit model.Objective To investigate the effect of telmisartan preconditioning on myocardial ischemia/reperfusion injury in a rabbit model.Methods Thirty male New Zealand white rabbits were randomly divided into three groups. The animal model grouping, the surgical procedure and the sample size were same to those in the Part One. The changes of hemodynamics were monitored continuously throughout the whole experiment. The values of HR, +dp/dtmax, -dp/dtmax and LVEDP were recorded at baseline, 1h after occlusion and 30min, 2h, 3h after reperfusion. Then, the animals were killed and infarct size of rabbit heart was determined by image analysis system after TTC staining. Tissue samples were removed from reperfusion zone. After disposal with routine histological methods and stained with hematoxylin-eosin (HE), the degree of injury in myocardium were examined by light microscopy. Apoptosis of cardiomyocytes was determined by transmission electron microscope (TEM). The apoptotic cells of ischemia zone are assessed by transferase-mediated dUTP nick end labeling (TUNEL) staining means. Result Compared with sham group, the values of HR, +dp/dtmax, - dp/dtmax were significantly declined and the values of LVEDP were significantly increased in model group and treated group at 1h after occlusion and 30min, 2h, 3h after reperfusion (P < 0.05). Compared with model group, the values of HR were restored better in treated group at 3h after reperfusion (P < 0.05), the values of +dp/dtmax were restored better in treated group at 2h and 3h after reperfusion (P < 0.05), the values of -dp/dtmax and LVEDP were restored better in treated group at 30min and 3h after reperfusion (P < 0.05). Compared with model group, treated group reduced the degree of injury, infarct size and apoptosis index of myocardium (P < 0.01). Telmisartan inhibited the morphological changes of apoptotic cardiomyocytes induced by myocardial ischemia/reperfusion such as cell atrophy, condensed chromatin clumps against the nuclear envelope and presentation of apoptotic body.Conclusion The telmisartan preconditioning plays a protective role in myocardial ischemia/reperfusion injury in the rabbit model by reducing the apoptosis of myocardium, diminishing the infarct size, lightening the degree of injury and improving the functional disturbance of left ventricle.