Role And Mechanism Of MAPK Family In Delayed Phase Of Isoflurane Induced Preconditioning On Rat Heart

Role and mechanism of MAPK family in delayed phase of isoflurane preconditioning on rat heartIschemic preconditioning had strong protective effect on myocardium, but its clinical use was limited. Drug preconditioning had more recognition not only because of its similar effect of myocardial protection like ischemic preconditioning but also because of its convenience, easy controlling and safety. Volatile anesthetics not only have myocardial protective effect on early phase but also have delayed myocardial protective effect on rats, rabbits and other animals. The mechanism involves many substances and signal transduction pathways such as: nitric oxide (NO), K_ATP channels, G protein-coupled receptors, protein kinase C, protein tyrosine kinase, mitogen-activated protein kinase (MAPK). It was found that MAPK were involved in early phase of myocardial protection preconditioned with volatile anesthetics, yet there was no report about its role on delayed phase of myocardial protection.ObjectsUsing isolated rat heart model perfused with Langendorff apparatus, this experiment was divided into two parts. Part 1: To investigate the action of different concentration of isoflurane induced delayed preconditioning on ischemic-reperfusion rat hearts in vitro and to explore the best concentration and the best interval time with isoflurane delayed preconditioning. Part 2: To investigate the expression and action of ERK, p38MAPK and JNK in ischemic-reperfusion heart preconditioned with isoflurane, then to investigate whether the effects can be antagonized or imitated using selective inhibitors of ERK, p38MAPK and JNK; To observe the content change of c-los mRNA, c-jun mRNA, HSP27 mRNA, which were the downstream substances of the three kinases and further to discuss the action and mechanism of MAPK family on rat myocardial protection with isoflurane induced delayed preconditioning. Materials1. Experimental animalsSprague-Dawley(SD) rats, regardless of sex, provided by Xuzhou medical college Laboratory Animal Center, weighed between 250-300g.2. Experimental reagentCalcium chloride(Xu Zhou second chemical agent factory), glucose(Shanghai rainbow light chemical plant), Potassium Chloride( first chemical agent factory in Jiangsu province), sodium bicarbonate(Shanghai rainbow light chemical plant), sodium chloride (Shanghai rainbow light chemical plant), potassium dihydrogen phosphate (Shanghai second chemical agent factory), Magnesium Sulfate(Lianyungang chemical agent plant), disodium ethylenediamine tetraacetic acid(EDTA) (Nanjing chemical agent plant), isoflurane(Abbott Laboratories, USA), triphenyltetrazolium chloride (Sigma, USA), CK kit(Nanjing), LDH kit(Nanjing), p-JNK antibody(G-7): sc-6254 (Santa Cruz Biotechnology, Inc ) , p-ERK antibody(E-4):sc-7383 (Santa Cruz Biotechnology, Inc), p-p38MAPK antibody(Santa Cruz Biotechnology,Inc), JNK antibody (Santa Cruz Biotechnology, Inc), ERK antibody(Santa Cruz Biotechnology, Inc), p-38MAPK antibody(Santa Cruz Biotechnology,Inc).3. Experimental instrumentLangendorff apparatus(USA); HL-4 Precision Pump(Shanghai); UV-240PC Ultraviolet Spectrophotometer(Japan); Photoelectrical Analytical Balance(Shanghai); BME33 Blood Gas Analysis Instrument((Danmark); SPECTRAMED Rheodynamic Monitor(USA); IVY401 Monitor(USA); HSS-1 Digital Ultra Constant Temperature Bath(Chengdu); Varian 3400 Gas Chromatography Instrument(USA); NORMAC Anesthetic Gas Monitor(Holand); Isoflurane Evaporator(Muraco medical Co. ltd); Olympus AU 1000 Auto Biochemical Analyzer(Japan); Imagemaster VSD Graph Analyzer(USA); Maclab/4 modulus convertor(Australia); Apple Macintosh 7200/90 Computor(USA); RS-3200 Pressure Transducer(Australia); CR-21 High-speed frosted centrifuge(Japan); HARRIS ultra low temperature refrigerator (USA).Methods1. The effect of isoflurane induced delayed preconditioning on myocardial ischemia-reperfusion injury. 104 SD rats were randomly divided into 13 groups(n=8 in each group). Control group(Con): Using langendorff apparatus, the myocardial ischemia-reperfusion injury model were made in isolated rat hearts, the hearts were ischemia for 30min followed by 120min reperfusion; Oxygen preconditioning with 24h interval group(O₂24h); Oxygen preconditioning with 48h interval group(O₂48h); Oxygen preconditioning with 72h interval group(O₂72h); In these three groups, rats were inhaled pure oxygen for 2h. 0.75MACisoflurane preconditioning with 24h interval group(0.75Iso24h); 0.75MAC isoflurane preconditioning with 48h interval group(0.75Iso48h); 0.75MAC isoflurane preconditioning with 72h interval group(0.75Iso72h); In these three groups, the rats were inhaled mixture of oxygen and 0.75 MACisoflurane for 2h. 1.0 MAC isoflurane preconditioning with 24h interval group(1.0 Iso24h); 1.0 MAC isoflurane preconditioning with 48h interval group(1.0 Iso48h); 1.0 MAC isoflurane preconditioning with 72h interval group(1.0 Iso72h); In these three groups, the rats were inhaled mixture of oxygen and 1.0 MAC isoflurane for 2h. 1.5 MAC isoflurane preconditioning with 24h interval group(1.5 Iso24h); 1.5 MAC isoflurane preconditioning with 48h interval group(1.5 Iso48h); 1.5 MAC isoflurane preconditioning with 72h interval group(1.5 Iso72h); In these three groups, the rats were inhaled mixture of oxygen and 1.5 MAC isoflurane for 2h. After preconditioning in above 9 groups, the rats were put into air incubator for 24h, 48h or 72h respectively( the rectal temperature were kept in 37℃), then using langendorff perfusion system, the ischemic-reperfusion model with ordinary temperature in isolated rat heart were made, with 30min ischemia followed by 120min reperfusion. Using Maclab/4s experimental system, LVSP, LVEDP, LVDP, +dp/dt max, -dp/dt max and HR were recorded at the end of equilibration, immediately after reperfusion and at 1 min, 3 min, 5 min, 10 min, 20 min, 30 min, 60 min, 90 min and 120min after reperfusion; then using chemical method, CK and LDH in coronary artery effluent were measured at the end of equilibration, 1min, 5min, 10 min, 20 min, 30 min, 60 min, 90 min and 120min after reperfusion and myocardial infarction size in left ventricular were measured using TTC staining.2. The effect and mechanism of MAPK family in delayed phase of myocardial protection preconditioned with isoflurane. According to the result of part 1, 1.5MAC isoflurane preconditioning with 48h interval had the maximal myocardial protective effect. 56 rats were randomly divided into 7 groups(n=8 in each group). Control group(Con); 1.5MAC isoflurane preconditioning with 48h interval group(1.5 Iso 48h); these two groups were managed as in Part 1. DMSO+1.5MAC isoflurane preconditioning with 48h interval group(DM group); PD98059+1.5MAC isoflurane preconditioning with 48h interval group(PD group); SB203850+1.5MAC isoflurane preconditioning with 48h interval group(SB group); SP600125+1.5MAC isoflurane preconditioning with 48h interval group(SP group); SP600125+control group(SP+Con group). The rats in above 5 groups were intraperitoneal injected with 10ï¼…DMSO 1ml, PD98059 0.5mg/kg( ERK inhibitor), SB203580 1mg/kg(p38MAPK inhibitor), SP600125 20mg/kg(JNK inhibitor)and SP600125 20mg/kg respectively 45min before ischemia. The heart model, method and index of measurement were the same as in part 1. Besides, part of myocardium of left ventricular apex were cut down for two purpose: one part was made as paraffin section and to detect the phosphorylation change of ERK, p38MAPK and JNK using immunohistochemical method; the other part was refrigerated with liquid nitrogen for measuring the content of c-fos, c-jun and HSP27mRNA using RT-PCR and semiquantitative analyzing the activity of ERK,p38MAPK,JNK using western blot later.ResultsPart OneThe cardiac function indexes had no significant difference(P＞0.05) at the end of equilibration in all groups. LVEDP was significantly lower during reperfusion period in all isoflurane preconditioning groups than in control group and in oxygen preconditioning groups(P＜0.05, P＜0.01); LVEDP at 3 min and 5min during reperfusion in 1.5Iso48h group was significantly lower than in three 0.75Iso groups (P＜0.05); The recovery rate of LVDP in each isoflurane preconditioning group was higher than in control and oxygen preconditioning groups, especially higher at 30min during reperfusion in 1.5Iso48h group(63ï¼…)than in control group(39ï¼…). The recovery rate of +dp/dt max at 20 min,30 min and 60min during reperfusion in 1.5Iso groups and in 1.0Iso groups was higher than in control group(P＜0.05, P＜0.01); +dp/dt max at 60min, 90min, 120 min during reperfusion in 1.5Iso48h group was still higher than in control group(P＜0.01) and at 20min, 30min, 45 min was higher than in oxygen preconditioning group(P＜0.05, P＜0.01); -dp/dt max at 30 min, 45 min, 60 min, 90 min and 120min during reperfusion in 1.5Iso48h group was significantly higher than that of control group, oxygen preconditioning group and 0.75IsoP group (P＜0.05, P＜0.01); and at 20 min, 30min was significantly higher than that of 1.0Iso24h group(P＜0.05), and at 20 min higher than 1.0Iso72h group(P＜0.05). The HR at 20 min, 30 min, 45 min, 60 min, 90 min and 120min during reperfusion in 1.5Iso48h group was significantly higher than in control group and O₂ preconditioning group(P＜0.05, P＜0.01); and at 30 min, 45 min during reperfusion in 1.5Iso24h group, 1.5Iso72h group, 1.0Iso48h group and 1.0Iso72h group HR was higher than in control group(P＜0.05, P＜0.01); The above cardiac function indexes in 0.75Iso preconditioning group were better than in control group, but had no statistically significance. The release of CK and LDH after reperfusion in each isoflurane preconditioning group was significantly lower than in control and oxygen preconditioning group(P＜0.05, P＜0.01); The release of LDH at all time points during reperfusion in 1.5Iso48h group was lower than in 0.75 MAC isoflurane preconditioning groups and oxygen preconditioning groups(P＜0.05, P＜0.01), and at 60 min, 90 min and 120min during reperfusion in 1.5Iso48h group release of LDH was lower than in 1.0Iso24h group and 1.0Iso72h group(P＜0.05, P＜0.01). The release of LDH at 5min, 10min, 20min, 90 min and 120min during reperfusion in 1.5Iso48h group was significantly lower than that in 0.75M isoflurane preconditioning group(P＜0.05, P＜0.01), and at 20min was lower than in1.0Iso24h group and 1.5Iso24h group(P＜0.05, P＜0.01). The infarct size in each isoflurane preconditioning group was significantly smaller than in control group(P＜0.05, P＜0.01); the infarct size in 1.5Iso48h group and 1.0Iso48h group was smaller than in all 0.75Iso groups and O₂ preconditioning groups(P＜0.01).Part Two1. PD group: The cardiac function index exacerbated significantly, the release of CK and LDH and the infarct size increased significantly in PD group compared with 1.5Iso48h group(P＜0.05, P＜0.01). PD98059, the ERK inhibitor, partly cancelled the protective effect of isoflurane preconditioning. Immunohistochemistry staining showed: ERK phosphorylation and nuclear translocation in control group had not been seen. Characteristic of activation and nuclear translocation of ERK which showing that brown masculine granulation dispersed in cytoplasm and enriched in nucleus had been found in myocardial cell in 1.5Iso48h group. Semiquantitative analysis of Western blot showed the expression amount of p-ERK1/2 in 1.5Iso48h group was significantly higher than in control group(P＜0.01). PD98059 suppressed the expression of p-ERK1/2 significantly. The results of RT-PCR showed content of c-fos mRNA in 1.5Iso48h group was much higher than in control group(P＜0.01); PD98059 reduced content of c-fos mRNA greatly(vs. 1.5Iso48h group, P＜0.01).2. SB group: Compared with 1.5Iso48h group, the recovery of cardiac function index decreased, and the release of CK and LDH and the infarct size increased significantly in SB group(P＜0.05, P＜0.01). SB203580, the inhibitor of p38MAPK, partly cancelled the protective effect of isoflurane preconditioning. Immunohistochemistry staining showed: p38MAPK phosphorylation and nuclear translocation in control group had not been seen. Characteristic of activation and nuclear translocation of p38MAPK which showing that brown masculine granulation dispersed in cytoplasm and enriched in nucleus had been found in myocardial cell in 1.5Iso48h group. Semiquantitative analysis of Western blot showed the expression amount of p-p38MAPK in 1.5Iso48h group was significantly higher than in control group(P＜0.01). SB203580 suppressed the expression of p-p38MAPK significantly. The results of RT-PCR showed content of HSP27 mRNA in 1.5Iso48h group was much higher than in control group(P＜0.01); SB203580 reduced content of HSP27 mRNA greatly(vs. 1.5Iso48h group, P＜0.01).3. SP+ 1.5Iso48h group: Compared with 1.5Iso48h group, the cardiac function index, the release of CK and LDH and the infarct size in SP+ 1.5Iso48h group had no significant difference(P＞0.05); Immunohistochemistry staining showed: JNK phosphorylation and nuclear translocation in control group had been seen which were significantly suppressed in 1.5Iso48h group and SP+ 1.5Iso48h group. Semiquantitative analysis of Western blot showed the expression amount of p-JNK1/2 in 1.5Iso48h group and SP+ 1.5Iso48h group was significantly lower than in control group(P＜0.01). The results of RT-PCR showed content of c-jun mRNA in control group was much higher than in 1.5Iso48h group and SP+ 1.5Iso48h group(P＜0.01); content of c-jun mRNA in SP+ 1.5Iso48h group reduced further compared with 1.5Iso48h group and had significant difference(P＜0.01).4. SP+ Con group: The recovery rate of cardiac function index increased, and the release of CK and LDH decreased and the infarct size decreased significantly compared with control group(P＜0.05), but the protective effect was weaker than in 1.5Iso48h group and SP+ 1.5Iso48h group(P＜0.05); The expression of p-JNK 1/2 and the content of c-jun mRNA were lower than in control group(P＜0.01), but had no significant difference compared with 1.5Iso48h group and SP+ 1.5Iso48h group(P＞0.05).DiscussionKerstin et al firstly discovered that volatile anesthetics could induce myocardial ischemic preconditioning effect in 1997, which was named as anesthetic induced preconditioning(APC). It was confirmed that halothane, isoflurane, sevoflurane and desflurane all had APC effect, further more all of them showed early phase and late phase(or delayed phase) of protection. The early phase of protection appeared in minutes after volatile anesthetic preconditioning and could continue for 2～3h. Delayed phase of protection appeared in 12～24h after preconditioning and could continue for 48～72h, thus the delayed phase of protection has greater clinical significance. The effect of myocardial preconditioning is a result of a serial cellular cascade reaction under stimulus of various stress things acting on hearts. Studies of the mechanisms of ischemic PC has led to a signaling paradigm that can be divided into three linear events: (â…°) triggers, factors released during the PC stimulus, initiate the protective process while (â…±) mediators are in action during the sustained ischaemia period, for example protein kinases which are responsible for integrating the signal and modulating the end-effectors. (â…²) end-effectors, which could confer myocardial protection. Although several triggers and their underlying pathways have been identified, the end-effector of protection remains elusive. MAPK is one of the highly conservative serine-threonine protein kinases and is intracellular signal transduction kinase which has intimate relationship with cell proliferation, differentiation and apoptosis. It has mainly three subsets in mammal cells: ERKs(extra cellular signal-regulated kinase), JNKs/SAPKs(c-Jun NH₂-terminal kinase/stress-activated protein kinase) and p38MAPK. The up-stream kinase was Ras/Raf-MEK₁/MEK₂, MEKKs-MKK₄/MKK₇ and MKK₃/MKK₆ respectively. The extra cellular signals that activate ERK are mainly the stimulus of mitosis such as growth factor which induced cell proliferation, differentiation and so on. Many extra cellular stimuli such as ischemia, hypoxia, stress, ultraviolet ray and cytokine can mainly activate the pathway of p38 MAPK and JNK, then to affect the biological behavior of cells and the function of tissues and organs under different pathologic condition. It was regarded that MAPKs play a key role in mediating cell's survival and apoptosis.The role of MAPK family in IPC is one of the important mechanisms of preconditioning. It was found that MAPK play important role in IPC. Little had done about the effect of MAPK on volatile anesthetic preconditioning. With isolated heart model, Da Silva found that isoflurane induced early phase of protection did not need the activation of ERK1/2 and p38MAPK on triggering period. This phenomenon was different from IPC. ERK1/2 was strongly activated during ischemia-reperfusion and ERK inhibitor PD98059 was used during reperfusion could antagonize the protective effect of isoflurane showing that ERK exert its effect as mediator in APC.It was found that volatile anesthetic preconditioning may play a second window protective effect in rat and rabbit models, also it was showed that ROS, COX-2 and K_ATP channel play important role in the protective effect. There had no report about the role of MAPK family in volatile anesthetic induced delayed preconditioning.In this experiment, using isoflurane induced delayed preconditioning on isolated rat heart, we found that 1.5MAC isoflurane preconditioning with 48h interval had the best myocardial protective effect. The recovery of cardiac function, the release of myocardial enzymes and the decrease of myocardial infarction size were all better than other preconditioning groups. Immunohistochemistry showed that isoflurane preconditioning can activate ERK and p38MAPK, and can make it nuclear translocation. isoflurane preconditioning can also inhibit JNK activation; and PD98059 and SB203580, when used before ischemia-reperfusion, can antagonize the protective effect of isoflurane preconditioning in different degree. SP600125 can partly simulate the protective effect of preconditioning. All these evidences showed that isoflurane induced delayed protective effect may be achieved through activating ERK and p38MAPK and inhibiting JNK. c-fos, c-jun and HSP27 were the down-stream substrate of ERK, JNK and p38MAPK respectively. The result of RT-PCR showed that isoflurane preconditioning may up-regulate the expression of c-fos mRNA, HSP27 mRNA and down-regulate c-jun mRNA. The content change of these three genes may be interdicted by the inhibitors of three kinases respectively.As was discussed above, ERK, JNK and p38MAPK all take part in the delayed phase protection of isoflurane preconditioning. Maybe they play their role through regulating the expression of down-stream substrates c-fos, c-jun and HSP27.ConclusionsThrough this experiment we concluded that:1. The delayed myocardial protective effect of isoflurane preconditioning was dose-dependent, and also has relation with interval. 1.5MAC isoflurane preconditioning with 48h interval had the best protective effect.2. Isoflurane preconditioning may activate ERK and p38MAPK and inhibit JNK. The inhibitor of ERK and p38MAPK may antagonize the effect of isoflurane preconditioning in different degree while the inhibitor of JNK can partly simulate the protective effect.3. All of ERK, JNK and p38MAPK may take part in the delayed phase protection of isoflurane preconditioning, maybe through regulating the expression of down-stream substrates c-fos, c-jun and HSP27.