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ATP Sensitive Potassium Channel Activation by Non-hypotensive Doses of Nicorandil Results in the Effects in a Rabbit in Vivo Ameliorated Model of Myocardial Ischemia/reperfusion

Author: LuoYouJun
Tutor: DongZhenMing
School: Hebei Medical University
Course: Anesthesiology
Keywords: Reperfusion injury Apoptosis Mitochondrial membrane potential Sarcolemmal membrane permeability Phosphatidylserine externalized
CLC: R541
Type: Master's thesis
Year: 2005
Downloads: 93
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Abstract

Objective: This present study aimed to invetigate ATP sensitive potassium channel activation by non-hypotensive doses of nicorandil results in the effects rabbit in vivo ameliorated model of myocardial ischemia/reperfusion. Methods: Experiment protocol: Sixty-four healthy male New Zealand white rabbits weighing 2.0-2.5 kg, aged 4 months were randomly allocated to eight groups with 8 rabbits in each group: （1） Sham group, （2） Ischemia/reperfusion group （MIRI）, （3） nicorandil group （Nic, a non-selective ATP-sensitive potassium channel agonist that activates the sarcolemmal or mitochondrial ATP-sensitive potassium channels）, （4） nicorandil and 5-hydroxydecanoic acid group （Nic+5-HD, a selective mitochondrial ATP-sensitive potassium channel antagonist）, （5） nicorandil and glibenclamide group （Nic+Gli, Gli , a non-selective ATP-sensitive potassium channel antagonist that inhibition of sarcolemmal or mitochondrial ATP-sensitive potassium channels）. （6） Ischemic preconditioning（IPC） group,（7） IPC and 5-hydroxydecanoic acid group （IPC+5-HD）, （8） IPC and glibenclamide group （IPC+Gli）. A rabbit in vivo ameliorated model of myocardial ischemia/reperfusion was created, the details as follow: the white rabbits were anesthetized by intravenous injection of soluble pentobarbitone （30mg/kg）. The New Zealand rabbit subjected to 30 minutes of complete occlusion of the left circumflex coronary artery and 120 min of reflow. The trachea was incised and intubated autonomous breathing kept. Catheters were inserted into the femoral artery to collect blood sample and monitor arterial pressure. Electrodes were placed on paws to record electrocardiogram （ECG） and heart rate. A left thoracotomy was performed at the second-third intercostal space and the heart was exposed. The left circumflex coronary artery was occluded by snaring with a small tube through which the ligature had been passed. Ischemia was confirmed by ST segment alterations and cyanosis on the myocardial surface. After 30 minutes of occlusion （ischemia）, the ligature was loosened and the myocardial tissue was reperfused 120 minutes. Reperfusion was confirmed by the disappearance of cyanosis. The suture used to occlude the artery was left in place to localize the site of occlusion and to assess infarct size later. Sham group rabbits thoracotomy was performed but no ischemia. In MIRI group, the left circumflex coronary artery was occluded during 30 minutes and then reperfused during 120 minutes. In Nic group, early intravenous infusion of nicorandil （100 micro g/kg bolus+10 micro g/kg/min） before the coronaryocclusion, both in Groups （Nic+5-HD） and （Nic+Gli）, procedure similar to Nic group, 5-HD （5 mg/kg, i.v. bolus） or glibenclamide （5mg/kg, i.v. bolus） were injected 20 min before pharmacological preconditioning induced by nicorandil. In IPC group, IPC was elicited with four cycles of 5 minutes of the left circumflex coronary artery occlusion followed by 5 minutes of reperfusion before the 30 minutes occlusion period.both in Groups （IPC+5-HD） and （IPC+Gli）, procedure similar to IPC group, 5-HD （5 mg/kg, i.v. bolus） or glibenclamide （5mg/kg, i.v. bolus） were injected 20 min before IPC. Blood sample was taken from femoral artery after catheter was inserted into the femoral artery （T1）, left thoracotomy was performed （T2） the silk suture was located （T3）, the left circumflex coronary artery was occluded 30 minutes （T4） and reperfused 120 minutes （T5） for assaied of the concentration of maleic dialdehyde （MDA） with the method of Thibarbituric Acid, the activity of superoxide dismutase （SOD） with xanthine oxidase, the level of Troponin I （cTnI） with immunometic assay. Measurement of infarction: At the end of the infarction protocol, the ligature around of the left circumflex coronary artery was retightened and 10 ml of 1% Evans blue dye was injected as a bolus into the left atrium until the eyes turned blue. The animals were euthanized immediately, and the heart was removed and frozen. The heart was then cut from apex to base into five transverse slices of equal thickness. The area at risk was determined by negative staining with Evans blue.The slices were then incubated in 1% triphenyltetrazolium chloride solution in isotonic pH 7.4-phosphate buffer at 37°C for 20 minutes. Red-stained viable tissue was easily distinguished from the infarcted pale or unstained necrotic tissue. The areas of infarcted tissue, the ischemic risk zone, and the whole left ventricle were determined by computer morphometry with imaging software （Image J 1.33u, National Istitutes of Health ）. The area for each region was averaged from slices. Infarct size was expressed as a percentage of the ischemic risk area. Myocytes were stained with the use of markers of sarcolemmal membrane permeability: the fluorescence dyestuff Hoechst 33342 and propidium iodide were used. The different fluorescence was monitored with fluorescence microscope. The excitation wavelength of Hoechst 33342 was 352nm, and the emission fluorescence was monitored at 400⁵00nm. The excitation wavelength of propidium iodide was 488nm, and the emission fluorescence was monitored at 630nm. Hoechst 33342 stained the apototic myocytes; the necrotic myocytes were stained by propidium iodide. The optical images were obtained and inputed to a computer. For quantification of Hoechst33342 positive （apototic myocytes） and propidium iodide positive （necrotic myocytes）, Myocytes were counted regardless of morphology per field of vision（ ×200）. Quantitative image analysis was performed using image analysis software （ImageJ）.Myocytes were stained with fluorescein isothiocyanate （FITC）, annexin V, and propidium iodide were monitored with flow cytometry. AnnexinV binds phosphatidylserine （PS）, a phosphoaminolipid was externalized during apoptosis. Propidium iodide is a marker of cell necrosis. The changes in mitochondrial membrane potential （ m） were monitored using flow cytometry （EPICS-XL, Beckman Coulter） analysis. Myocytes analyzed using flow cytometry of （10 000 cells/sample） after loading of the dyes. The excitation wavelength was 488 nm, and the emission fluorescence for FITC was monitored at 530 nm （FL-1） and and the emission fluorescence for propidium iodide 630 nm （FL-2）. Therefore, The myocytes were divided four clusters injured myocytes （AnnexinV-PI+）, normal myocytes （AnnexinV-and PI-） necrotic myocytes （AnnexinV+ PI+）, apoptotic myocytes （AnnexinV+ PI-）. The flow cytometry data were analyzed using Cell Quest （Becton Dickinson）. The quantification of myocytes in different groups was counted and analysized. Assessment of mitochondrial membrane potential （ m） flow cytometry （EPICS-XL, Beckman Coulter） was used. Myocytes were stained with the dye 5, 5’, 6, 6’-tetrachloro-1, 1’, 3, 3’-tetraethylbenzimidazolcarbocyanine iodide （JC-1）. The changes in m were monitored with the dye JC-1. The m was monitored using flow cytometry （EPICS-XL, Beckman Coulter） analysis. Myocytes analyzed using flow cytometry of （10 000 cells/sample） after loading of the dye. The excitationwavelength was 488 nm, and the emission fluorescence for JC-1 was monitored at 530 nm （FL-1） and 582 nm （FL-2）. The flow cytometry data were analyzed using Cell Quest （Becton Dickinson）. The green fluorescence intensity and red fluorescence intensity were collected. The ratio of JC-1 aggregate intensity （red fluorescence intensity） to monomer intensity （green fluorescence intensity） was calculated. An increase in this ratio was interpreted as gain of m, whereas a decrease in the ratio was interpreted as m loss. Observation of myocardial ultrastructure with electron microscope: At the end of the infarction protocol, the animals were euthanized immediately, and the heart was removed. The heart was then cut open from base to apex. rinsed of excess blood.The myocardial tissue size of 1 mm3 was cut from the border of infarction along vertical axes,The myocardial tissues were subsequently fixed in 4% glutaralin solution for 6 hours.All the operations wre performed on ice and then the myocardial tissues were stored at 4°C at least 2 hours until used. The specimen observed with transmission electron microscope （H-7500）（electron microscope laboratory of Hebei Medical University）. Immunohistochemistry: The expression of Bcl-2 and Bax protein, cytochrome C was released from mitochondria into the cytoplasm. The release and distribution of cytochrome C in intact myocytes were assayed by the method ofimmunohistochemistry. For Caspase-3 staining, rabbit polyclonal antiserum raised against the activated form of Caspase-3, which recognizes only the processed 20-kDa subunit of cleaved Caspase-3, was used. The optical images were obtained at the same resolution rrightness, contrast, color saturation and same size per field of vision（ ×400）. The integraloptical density of these images was analysized using image analysis software （Motic 6.0）. The Ratio of Bcl-2 and Bax integraloptical density were calculated. Results: 1 The electrocardiogram （ECG） alteration before and after ischemia/reperfusion. Ischemia was confirmed by ST segment alterations:The ECG has no change before the left circumflex coronary artery was occluded. The ST segment elevation upward and the change of ST segment did not complete recover when the ligature loosened. 2 The change of SOD activity,MDA concentration and cTnI level: 2.1 At the different time points in the same group,there were no significant differences at T₁ and T₂～T₅ in Sham group（P>0.05）.Compared with T₁,there were no significant differences between T₁ and T₂～T₃ in other groups（P>0.05）,but there were significant differences between T₁ and T₄～T₅. The SOD activity was lower （P<0.05）,the MDA concentration and cTnI level were higher（P<0.05） in other groups. 2.2 At the same different time points in the different groups, there were no significant differences at T₁,T₂ and T₃ in all the groups（P>0.05）. Compared with Sham group at T₄and T₅,the SOD activity was decreased, MDA concentration and cTnI level were increased in MIRI group, Nic group,IPC group, Nic+5-HD group, Nic+Gli group, IPC+5-HD group and IPC+Gli group （ P<0.05 ） .There were no significant differences in other groups such as Nic+5-HD group, Nic+Gli group, IPC+5-HD group and IPC+Gli group （ P>0.05 ） . Compared with MIRI group at T₄ and T₅, The SOD activity was higher（P<0.05）, MDA concentration and cTnI level were lower（P<0.05）in Nic group and in IPC group（P<0.05）. but there was no significant differences in the other groups（P>0.05）. There was no significant differences between Nic group and IPC group（P>0.05）. Compared with Nic group at T₄ and T₅, the SOD activity was lower（P<0.05）, MDA concentration and cTnI level were higher （P<0.05）in the other groups. Compared with Nic +5-HD group at T₄ and T₅, the SOD activity was higher（P<0.05）, MDA concentration and cTnI level was lower（P<0.05）in IPC group, and there was no significant differences in Nic+Gli group,IPC+5-HD group and IPC+Gli group （P>0.05）.Compared with Nic+Gli group at T₄ and T₅, the compared results were as same as those of Nic+5-Hd group. Compared with IPC group at T₄ and T₅, the SOD activity was lower（P<0.05）,MDA concentration and cTnI level were higher （P<0.05）in IPC+5-HD group and IPC+Gli group. There was no significant differences between IPC+5-HD group and IPC+Gli group（P>0.05）. 3 Compared with MIRI group, nicorandil preconditioningor ichemic preconditioning markedly limits myocardial infarct size （P>0.05）, 5-hydroxydecanoate acid or glibenclamide abolished protection of nicorandil preconditioning, ischemic preconditioning （P<0.05）. The differences about the abolishment of 5-hydroxydecanoate acid or glibenclamide had no significant （P>0.05）. 4 There were a few Hoechst33342-positive cells （apototic cells） and propidium iodide positive cells （necrotic cells） in Sham group, Nic group and IPC group. There were a lot of Hoechst33342-positive cells and propidium iodide positive cells in MIRI group and the other block groups. 4.1 The statistical analysis results about count Hoechst33342-positive cells in all the groups were as follows: Compared with Sham group, the count of Hoechst33342-positive cells was increased in all the groups （P<0.05）. Compared with MIRI group, the count of Hoechst33342-positive was lower in Nic group and IPC group （P<0.05）,and there were no significant differences among the other block groups（P>0.05）. Compared with Nic group, there were no significant differences betwwen Nic group and IPC group（P>0.05）, 5-HD and Gli aboblished the protective effects of Nic pretreament and IPC（P<0.05）,and the aboblishment of 5-HD and Gli had no significant differences（P>0.05）.4.2 The statistical analysis results about count of propidium iodide positive cells in all the groups were as follows: There were no significant differences between Sham group and IPC group （P>0.05）, The statistical analysis results about count ofpropidium iodide positive cells in the other groups were as same as those of Hoechst33342-positive cells. 5 Results of flow cytometry analysis with annexin V-propidium iodide staining to examine apoptosis and necrosis: Quantification of cells of apoptosis and necrosis were mentioned mainly in different treatments here. Compared with Sham group, the cells of necrosis（Annexin V⁺ PI⁺） and of apoptosis（Annexin V+ PI-）in other groups are higher（P<0.05） but IPC group （P>0.05）. Compared with MIRI group, nicorandil preconditioning and ischemic preconditioning decreased the cells of necrosis and of apoptosis （P<0.05）. Nicorandil preconditioning had the protective effect as similar as ischemic preconditioning （P>0.05）. Both 5-hydroxydecanoate acid acd and glibenclamide prevented protection of nicorandil preconditioning and ischemic preconditioning （P<0.05）. The difference about abolishment of 5-hydroxydecanoate acid or glibenclamide had no significant difference （P>0.05）. 6 The ratio of JC-1 aggregate to monomer intensity changes in various groups. Compared with Sham group, green fluorescence intensity did not change significant difference among groups except （Nic+Gli） group（P<0.05）. Compared with Sham group, the ratio of JC-1 aggregate to monomer intensity was lower in all the groups（P<0.05）. Compared with MIRI group, the ratio of JC-1 aggregate to monomer intensity in Nic group and in IPC group was higher （P<0.05）. There was no significant differences between Nic group andIPC group（P >0.05）. Both 5-hydroxydecanoate acid and glibenclamide prevented protection of nicorandil preconditioning and ischemic preconditioning （P<0.05）. The difference about abolishment of 5-hydroxydecanoate acid or glibenclamide had no significant difference （P>0.05）. 7 The result of transmission electron micotelecope: Ultrastructural changes in various groups. Sham group, myoccardium had well-preserved ultrastructure. The myocardial myofilament arrayed in order. Chromatin material in nucleus was uniformly dispersed. Mitochondria （m） were elongated or oval in shape and abundant glycogen was observed. Myoccardium in MIRI group, （Nic+5-HD） group, （ Nic+Gli ） group, （IPC+5-HD） group and （IPC+Gli） group were broken and distorted. Myocytes showed clumped chromatin material in the nucleus, mitochondrial cristae were disrupted, and electron dense deposits were observed within mitochondria. Glycogen was reduced. Myocardial myofilament arrayed in order, the nucleus and mitochondria were well-preserved, and there were abundant glycogen in mitochondria in IPC group and Nic group. 8 The changes of integraloptical density of cytC,activated Caspase-3, Bcl-2 protein,Bax protein and the ratio of Bcl-2 and Bax integraloptical density: 8.1 the ratio of Bcl-2 and Bax integraloptical density: Compared with Sham group, there were no significant difference in IPC group and Nic group （ P >0.05）, the ratio of Bcl-2 and Bax integraloptical density were lower in the other groups（P <0.05）.Compared with MIRI group,the ratio of Bcl-2 and Bax integraloptical density was increased in IPC group and in Nic group. 5-hydroxydecanoate acid or glibenclamide prevented protection of nicorandil preconditioning and ischemic preconditioning （P<0.05）. The difference about abolishment of 5-hydroxydecanoate acid or glibenclamide had no significant （P>0.05）. 8.2 The statistical analysis results about the changes of integraloptical density of cytC and activated form of Caspase-3 staining in cytoplasm were as same as those of Bcl-2 and Bax integraloptical density. Conclusions: 1. Non-hypotensive doses of nicorandil pretreatment minish myocardial infarct size in a rabbit in vivo ameliorated model of myocardial ischemia/reperfusion through reducing ischemia/reperfusion-induced myocardial apoptosis. 2. The expression of the Bcl-2 is increased; overexpression of the Bax is inhibited. Cytochrome C translocation to the cytosol and activation of Caspase-3 were inhibited by nicorandil pretreatment. Nicorandil pretreatment inhibits cardiac myocytes of apoptosis. 3.The protective effects of non-hypotensive doses of nicorandil pretreatment are mediated by activation of ATP sensitive potassium channel channels, especially or mainly those of mitochondrial ATP-sensitive potassium channels. 4. Mitochondria are the critical organelle for myocyte cell survival. Mitochondria play a key role in the signal transduction pathways mediating the happen of apoptosis or necrosis.

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