The Study On Effects And Mechanisms Of The Non-Invasive Limb Ischemic Preconditioning On Ischemic Ventricular Arrhythmias In Rabbits

Abstract:Objective:To study on effects and mechanisms of the non-invasive limb ischemic preconditioning on ischemic ventricular arrhythmias in rabbits thus provides a practically theoretical basis for its clinical trial.Methods:Twenty-seven rabbits had been randomly divided into the control group(Ctr, n=8),non-invasive limb ischemic precondi-tioning group(LIP, n=7), LIP+ glibenclamide group(LG, n=6) and LIP+ SB203580 group(LSB,n=6).After anesthesia, the right external jugular vein was dissected and cannulated to the right ventricle to record the endocardium MAP. Needle electrodes were punctured in four limbs of the rabbit to record electrocardiogram (ECG).The contact electrode was inserted from the 4th or 5th ribs beside the left sternal to record the epi-cardium monophasic action potential(MAP).Ctr group does not been given any special treatment, but the vasopressin (2U/kg) was injected intravenously to produce myocardial ischemia background, then epine-phrine (0.2ml/kg) was injected to induce arrhythmia, when the records of ECG, endocardium and epicardium MAP were stabilized.The ECG, endo-cardium and epicardium MAP were simultaneously recorded, and the changes of endocardium & epicardium MAP and the relationship to arr- hythmia were observed.LIP group:pneumatic tourniquet was used to occlude the blood flow of rabbits'two hind limbs make them ischemia for 5 minutes,then loosened to reperfusion for 5 minutes,and repeated it for four times.Another methods and contents of observation were same as the Ctr group.LG group:Firstly, glibenclamide(0.16mg/kg) was intra-venous injection,30 minutes later, to carry out the same treatments as the LIP group.LSB group:After the SB203580 (0.1mg/kg) was intravenous injection for 30 minutes,to carry out the same treatment as the LIP group. ECG, the changes of endocardium & epicardium MAP, the types,inci-dence and duration of arrhythmia were simultaneously recorded.At the end of the experiment, air embolism was used to kill the animals, then remove the heart, and some parts were fixed with 4% paraformaldehyde for morphological observation, the other parts were stored in-70℃refri-gerator for another detections.Results:①Before and after ischemia, endocardium APD90 were(130.66±13.36)ms and(135.23±10.24)ms, epicardium APD90 were(132.56±16.27)ms and(141.90±16.37)ms in the Ctr group.Endocardium APD90 were(122.47±13.94)ms and(130.26±17.12)ms, epicardium APD90 were(124.16±9.72)ms and(148.02±12.78)ms in the LIP group.Endocardium APD90 were(127.19±36.78)ms and (134.72±46.01)ms, epicardium APD90 were (146.20±16.21)ms and (160.78±22.73)ms in the LG group.Endocardium APD90 were(100.88±13.36)ms and(104.56±28.46)ms,epicardium APD90 were(105.77± 18.59)ms and(104.10±20.69)ms in the LSB group.After ischemia, all rabbits presented arrhythmia, mainly represented as frequent ventricular premature contraction of bigeminy or trigeminy. There were 3 cases of ventricular tachycardia and no ventricular fibrillation in the Ctr group. There was no ventricular tachycardia and ventricular fibrillation in the LIP, LG and LSB group.The duration of arrhythmia(s)was (231.54±15.78),(167.61±16.13),(159.38±29.56) and (209.83±26.39) respec-tively.②The heart tissue detection of Ctr, LIP, LG & LSB group respectively indicated,SOD activity (U/mgprot) was (263.78±61.51), (322.63±50.46),(280.56±37.01)and(314.52±44.72).MDA content (nmol/mgprot) was(14.01±6.23),(5.34±2.17),(6.25±2.57) and (5.94±2.13).NO content(μmol/gprot) was (30.85±10.13),(242.17±48.84), (68.18±31.26)and(65.18±22.55).③After ischemia,endocardium APD90 was extended in each group,but endocardium APD90 of LIP and LSB group were shorter than Ctr group(P<0.05).Epicardium APD90 of Ctr group, LIP and LG group were extended while LSB group was shortening, and epicardium APD90 of the LSB group was shorter than Ctr and LIP group(P<0.05).The duration of arrhythmia of the LIP and LG group was shorter than the Ctr group(P<0.05).SOD activity of LIP group was higher than Ctr group(P<0.05).SOD activity of LG and LSB group compared with Ctr and LIP group no significant difference(P>0.05).The MDA levels of LIP, LG and LSB group were lower than Ctr group (P<0.01),and the MDA content of LG and LSB group compared with LIP group no significant difference(P>0.05).The NO content of LIP group was higher than Ctr group(P<0.01),and the NO content of LG and LSB group were lower than LIP group(P<0.01).④The relative expres-sion of HSP70 mRNA was(3.04±0.20),(3.14±0.20),(2.65±0.18)and (2.61±0.31)respectively. The relative expression of LIP group HSP70 mRNA compared with the Ctr group no significant difference(P>0.05), while the relative expression of LG and LSB group HSP70 mRNA were lower than Ctr and LIP group(P<0.01).The relative expression of COX-2 mRNA was(1.36±0.16),(1.61±0.22),(1.47±0.19)and(1.14±0.09) respectively. The relative expression of LIP group COX-2 mRNA was higher than Ctr group(P<0.05),and the relative expression of LSB group COX-2 mRNA was lower than Ctr and LIP group(P<0.05),while the relative expression of LG group COX-2 mRNA compared with Ctr and LIP group no significant difference(P>0.05).The relative expres-sion of iNOS mRNA was(1.32±0.08),(1.75±0.12),(1.40±0.14)and(1.34±0.07) respectively. The relative expression of LIP group iNOS mRNA was higher than Ctr group(P<0.01),and the relative expression of LG and LSB group iNOS mRNA were lower than LIP group(P<0.01).The rela-tive expression of TNFa mRNA was(0.57±0.03),(0.38±0.03),(0.55 +0.06) and (0.54±0.04)respectively. The relative expression of LIP group TNFa mRNA was lower than Ctr group(P<0.01),and the relative expression of LG and LSB group TNFαmRNA were higher than LIP group(P<0.01).The relative expression of NF-κB mRNA was (0.62±0.05),(0.51±0.03),(0.59±0.04)and (0.60±0.04)respectively. The relative expression of LIP group NF-κB mRNA was lower than Ctr group(P<0.01),and the relative expression of LG and LSB group NF-κB mRNA were higher than LIP group(P<0.01).⑤The protein expression of p-p38MAPK was (0.08±0.02)in the Ctr group,(0.15±0.03)in the LIP group, (0.12±0.02)in the LG group, The protein expression of p-p38MAPK in the LIP and LG group were higher than Ctr group (P<0.05).While it was (0.05±0.02)in the LSB group,and lower than the Ctr, LIP and LG group(P<0.05).Conclusion:1.LIP can reduce the injury of ischemic myocardial oxidative and improve myocardial blood supply, thus enhance electrical stability of ischemia myocardium.2.p38MAPK plays a very important role on LIP increasing cardiac electrophysio-logical stability, but KATP may participate only in antioxidation and to improve the blood supply of myocardium.3.The possible mechanisms and pathways of signal transduction of LIP protecting the I/R myocar-dium as follow. At first, LIP produce the "triggering substances" to switch on myocardial protection, then through the "intermediary" to start signal transduction, after that, mitogen-activated protein kinase system was activated to induce the signal transmission and amplification, and the activation of nuclear transcription factors,then the production of "effect- or" educe the myocardial protection effects.