| Objectives:(1) To investigate the protection of Isoflurane preconditioning on myocardial ischemia reperfusion injury in human. (2) To investigate the changes of myocardial protein expression profiles after Isoflurane pretreatment, and to search for the proteins probably involved in the early phase of preconditioning induced by Isoflurane with proteomics techniques. (3)To identify some different proteins with western blot according the above results.Methods:The experiment consists of three parts:(1) Thirty patients undergoing elective cardiac valve replacement with cardiopulmonary by pass (CPB) were randomly assigned to control group (n=15) and isoflurane group (n=15). In the isoflurane group, isoflurane of 1.0 minimum alveolar concentration end-tidal (1.1%～1.2%) was administered for 30 min followed by a 15 min washout period before the CPB. The control group did not inhale isoflurane, and there was no difference in the other drugs in the 2 groups. Pulmonary arterial dates were measured before inhalation isoflurane (T0),30 min after reperfusion(T1), after closing of thorax(T2), and 6h(T3),12h(T4) and 24h(T5) after reperfusion, respectively. Blood samples for serum cTnI and CKMB levels were obtained at To, T1, T3, T4 and T5. Right atria biopsies were collected at the times when inserting catheter into superior vena cava (SVC) and pulling out the catheter from SVC,the tissue and cell injury of myocardium was examined with optical and electron microscope. (2) Proteomic analysis of myocardium of isoflurane pretreatment. The right atria biopsies of isoflurane group or control group were sampled for proteomic analysis. The total proteins were extracted and separated by two dimensional gel electrophoresis(2-DE), and 16 differential expression protein spots were analyzed with matrix-assisted laser desor-ption/ ionization time-of-flight mass spectrometry (MALDI-TOF-MS). (3) According the above results, some proteins were identified with western blot.Results (1) Comparing with To, a significant decrease of mean arterial blood pressure (MABP) was detected at T1～T4(P<0.05或P< 0.01), a significant decrease of mean pulmonary arterial pressure(PAOP) was detected at T2～T5 and systemic vascular resistance(SVR) were detected at T1～T5(P<0.05 or P<0.01), stroke volume(SV) and stroke index(SI) at T1～T5, and cardiac output(CO), cardiac index(CI) and left ventricular stroke work index(LVSWI) at T1～T5 increase significantly in both control and isoflurane group(P<0.05或P<0.01); Comparing with control group, PAOP decreased significantly at T5(P<0.05), and SV, SI at T4～T5, CO, CI and LVSWI at T3～T5 increased significantly(P<0.05 or P<0.01) in isoflurane group. There was a significant decrease of cTnI level on T3～T5 and CKMB level on T1～T5 in isoflurane group (P<0.05) when comparing to control group.The tissue and cell injury of myocardium examined with optical and electron microscope in isoflurane group was decreased compared with the control group. (2) Analysis of 2-DE showed that 417±15 protein spots were seen in control group and 454±19 protein spots in isoflurane group, and that the expression of 20 protein spots were different between the two groups.16 protein spots were chosen to do MS analysis, and 16 proteins were preliminarily identified. Six proteins were up-regulated and three proteins were down-regulated and one protein was appeared in isoflurane group, but ten proteins were disappeared in isoflurane group. These proteins can be classified into four functional groups:Include cytoskeletal protein (Alpha actin,Alpha-myosin heavy chain,mutant desmin,vimentin),metabolism related proteins (aldehyde reductase,glyceraldehyde-3-phosphate dehydrogenase,mitochondrial ATP synthase),Transcription associated protein (general transcription factor IIIC,transcription factor AP2,zinc finger protein 771),and ionophorous protein (transient receptor potential cation channel). (3)We further detected the expression of Alpha actin (200 spot),Alpha-myosin heavy chain(277 spot),vimentin (221 spot) and mitochondrial ATP synthase (47 spot) by using immunobltting (Western Blot). It was sure that Alpha actin,Alpha-myosin heavy chain,vimentin and mitochondrial ATP synthase were up-regulated by isoflurane preconditioning.Conclusions:(1) Isoflurane pretreatment can result in the changes of protein expression profiles in the myocardium. The differential proteins might function as decreas injury of cytoskeletal proteins and promote the energy metabolism and regulation of genes and proteins of myocardium to confer cardioprotection. Among these differential proteins, we firstly discovered that Alpha actin,Alpha-myosin heavy chain,mutant desmin,vimentin,aldehyde reductase,glyceraldehyde-3-phosphate dehydrogenase,mitochondrial ATP synthase,general transcription factorⅢC,transcription factor AP2,zinc finger protein 771,transient receptor potential cation channel were involved in the isoflurane-induced early phase of preconditioning. (2) It was sured that Alpha actin,Alpha-myosin heavy chain,vimentin and mitochondrial ATP synthase were promoted with early phase of isoflurane preconditioning by using Western Blot.
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