The Study Of Protecting Effects And Mechanisms Of Bisoprolol On Myocardial Injury By Ischemia-reperfusion In Cardiomyocytes

Objective:Myocardial ischemia reperfusion injury model in SD rats and primary culture cardiomyocyte model of hypoxia/reoxygenation were established to investigate the protecting effect of cardiomyocytes mitophagy pathway with bisoprolol postprocessing.autophagosome blockers chloroquine and decreased Drp1 expression level with lentivirus expressing Drpl-specific siRNAinterference were used to study the role of bisoprolol on Drpl/Pink 1/Parkin pathway,to investigate the protective effect of bisoprolol on I/R or H/R induced mitochondrial autophagy of cardiomyocytes.Methods:Part1:The protective effects of bisoprolol postprocessing on mitochondrial autophagy was invested by using the myocardial ischemia reperfusion in SD rats.Therats underwent an open left thoracotomy,60 min of LAD ligation and then 72 hour of reperfusion,the myocardial ischemia reperfusion injury model in SD rats was established.Rats were randomly assigned into five groups:Sham-operated group(Sham group,n=10),Mode group(ischemia reperfusion group,I/R group,n=10),chloroquine+ischemia reperfusion group(CQ+I/R group,n=10),ischemia reperfusion+bisoprolol postprocessing group(I/R+BS group,n=10),and chloroquine+ischemia reperfusion+bisoprolol postprocessing group(CQ+I/R+BS group,n=10).Cardiac function was detected in all groups by ultrasonography.To The pathological changes of the cardiac tissue was observed using HE staining.Myocardial cell apoptosis in SD rat cardiac tissue detected with a fluorescence microscopy was observed,Using immunofluorescent labeling with TUNEL,and Evans blue-TTC double staining was also used for infarction.The protein level of LC3、Beclin1、OPA1、Drp1、p-Drp1、Pink1 and Parkin was determined by Western Blotting.Part2:The protective effects of bisoprolol postprocessing on mitochondrial autophagy was investigated by using primary cardiomyocytes hypoxia reoxygenation.We established primary myocardial hypoxia/reoxygenation(6h/6h)model after neonatal SD rats’(1-3d)cardiomyocytes were isolated and cultured for 36h,primary culture cardiac myocyte were randomly assigned into seven groups:controlgroup(CTL group),bisoprolol group(BS group)、chloroquine group(CQ group)、hypoxia/reoxygenation group(H/R group)、hypoxia/reoxygenation+bisoprolol postprocessing group(H/R+BS group)、chloroquine+hypoxia/reoxygenation group(CQ+H/R group)、chloroquine+hypoxia/reoxygenation+bisoprolol postprocessing group(CQ+H/R+BS group),experimental repetition times n=3;Troponin T(cTnT)were used to identify the original purity of primary culture cardiac myocyte,cell apoptosis were evaluated by flow cytometry,the mitochondrial morphology and the mitophagosome in cardiac tissue were observed by using the transmission electron microscope,the protein level of LC3、Beclin1、OPA1、Drp1、p-Drp1、Pink1 and Parkin was determined by Western Blotting.Part3:Drpl low expression lentiviral vector(si-Drpl)as Constructed,primary myocardial cells were transfected by Lentivirus stock,Transfection rate of the protein level of Drpl and was determined by Western Blotting.Thenwe established Primary myocardial hypoxia/reoxygenation(6h/6h)model after myocardial cells were successfully transfected.The cell was randomly assigned into seven groups:Control group(CTL group),bisoprolol group(BS group)、chloroquine group(CQ group)、hypoxia/reoxygenation group(H/R group)、hypoxia/reoxygenation+bisoprolol postprocessing group(H/R+BS group)、chloroquine+hypoxia/reoxygenation group(CQ+H/R group)、chloroquine+hypoxia/reoxygenation+bisoprolol postprocessing group(CQ+H/R+BS group);experimental repetition times n=3;cell apoptosis rates were evaluated by flow cytometry,the protein level of LC3、Beclinl、Pink1 and Parkin was determined by Western Blotting.Result:1.Showed the degree of myocardial infarction by Evans blue and TTC double staining(AI/AAR):compared with I/R group,the myocardial infarction area of I/R+BS group was lightened and the difference had statistical significance(0.3 1 ±0.021vs.0.44±0.03,P<0.05;compared with CQ+I/R group,the myocardial infarction area of CQ+I/R+BS group was lightened and the difference had statistical significance(0.41±0.033 vs.0.57±0.040,P<0.05).2.Ultrasonic cardiogram:compared with I/R group,LVEF,FS of I/R+BS group increased(0.76±0.054vs.0.54±0.032,P<0.05;0.40±0.026vs.0.24±0.022,P<0.05),LVEDV,LVESV decreased(0.48±0.022vs.0.76±0.034,P<0.05;0.11±0.012vs.0.35±0.016,P<0.05);compared with CQ+I/R group,LVEF,FS of CQ+I/R+BS group increased(0.64±0.032vs.0.50±0.052,P<0.05;0.30±0.012vs.0.19±0.016,P<0.05),LVEDV,LVESV decreased(0.71±0.032vs.0.81±0.042,P<0.05;0.28±0.014vs.0.42±0.024,P<0.05).3.TUNEL staining:compared with I/R group,the apoptotic index of I/R+BS group decreased(15±1.0vs.27±1.2,P<0.05);compared with CQ+I/R group,the apoptotic index of CQ+I/R+BS group decreased(26±1.6vs.38±2.2,P<0.05).Flow cytometry:compared with H/R group,the apoptotic index of H/R+BS group decreased(p<0.05);compared with CQ+H/R group,the apoptotic index of CQ+H/R+BS group decreased(p<0.05).4.Western blotting:compared with I/R group,myocardial cell rate of LC3II/LC3I and OPA1/Beclinl/Parkin/Pink1 protein expression level of IR+BS group increased,and p-Drp1 expression level decreased(p<0.05).compared with CQ+IR group,myocardial cell rate of LC3II/LC3I and OPA1/Beclinl/Parkin/Pink1 protein expression level of CQ+IR+BS group increased,and p-Drp1 expression level decreased(p<0.05).compared with H/R group,myocardial cell rate of LC3II/LC3I and OPA1/Beclinl/Parkin/Pink1 protein expression level of HR+BS group increased,and p-Drp1 expression level decreased(p<0.05).compared with CQ+H/R group,myocardial cell rate of LC3II/LC3I and OPA1/Beclinl/Parkin/Pink1 protein expression level of CQ+HR+BS group increased,and p-Drp1 expression level decreased(p<0.05)5.Transmission electron microscope:after anoxia/reoxygenation,primary cardiac muscle mitochondria arranged in disorder,mitochondria was swelling,mitochondrial cristae was ruptured,mitochondrial autophagy decreased;compared with HR group,the mitochondria damage of bisoprolol treatment group was lightened and mitochondria autophagosome increased(p<0.05).6.Compared with empty carrier group,primary cardiac myocyte apoptosis induced by anoxia/reoxygenation in Drp1 low expression group was lightened and mitochondria autophagy related protein Pink1 protein expression amount increased(p<0.05);compared with Drpl empty carried group,bisoprolol further upregulated anoxia/reoxygenation induced myocardial cell Pink1 protein expression level in Drpl low expression group(p<0.05)Conclusion:In this study,we found that ischemia reperfusion or Hypoxia reoxygenation could induce myocardial cell injury,mitochondrial autophagy lever decrease,and bisoprolol could increase mitochondrial autophagy to reduce myocardial cell injury,its mechanism was involved in the activation of Drpl/Pink1/Parkin mitophagy pathway via reducing the phosphorylation of Serine 637 site in Drp1,increasing protein level of Pink1 and Parkin,and thus increasing mitochondrial autophagy and cell survival.But chloroquine pretreatment may partially attenuate the effect of bisoprolol.