Study On The Change And Role Of β3-adrenoceptor In Myocardial Depression Of Septic Shock Rats Induced By LPS

IntroductionSepsis is the systemic inflammatory response syndrome caused by infection,the incidence of severe sepsis and septic shock gradual rise in mortality of nearly 50%.Myocardial depression caused by ventricular dysfunction or failure is very common in severe sepsis,the severity of heart failure and extent of disease is closely related to mortality.However,the pathophysiology of myocardial depression in sepsis is not very clear,there is usually not severe myocardial necrosis in septic myocardial dysfunction,which suggesting that cardiac function in septic shock is a transient change in the main,so making sure of the pathophysiology of sepsis transient myocardial depression will help clinicians to find new therapeutic targets and reduce the mortality of critically ill patients.β-adrenergic receptor(β-AR)is an important substance regulating heart function,the incidence of myocardial depression in severe sepsis and septic shock is closely related to the development of β-AR signal transduction system changes.Normally,β1-AR expression is 70% ~ 80%,β2-AR 20% to 30%,and β3-AR is low,but there are significant changes in bulk density in pathological conditions(e.g.,heart failure),researchers found that and protein expression of β3-AR is rising in patients with myocardial ischemia and ventricular dilatation,cardiac β3-AR expression also appeared in other pathological conditions.β3-AR activation requires more catecholamines than β1-AR and β2-AR activation,there are high tissue or circulating catecholamine levels to activate the β3-AR under pathological conditions.The patients of severe sepsis and septic shock had significantly higher serum concentrations of catecholamines,which is due to not only endogenous catecholamines produced by the patient’s own,but also the therapeutic intervention of exogenous catecholamines adrenaline.What is the role of the increasing of β3-AR in myocardial depression in sepsis shock is unclear,a high concentration of catecholamines compensatory protective response or septic myocardial depression promoting factor?Many studies have proven that exciting β3-AR will produce negative inotropic effect no matter the level of myocardial cells or in the heart,by activating inhibitory G protein(Gi protein),which in turn increase nitric oxide(NO)release,and NOS / NO-cGMP-PKG may be the downstream signaling transduction pathway of β3-AR,and may be closed to L-type calcium channel inhibition and intracellular calcium transient amplitude.It has been proved in the heart,there are three NOS isoforms,endothelial NOS(eNOS,NOS3),neuronal NOS(nNOS,NOS1)and inducible NOS(iNOS,NOS2).NOS1 and NOS3 functional express in the heart,but NOS2 express in pathological states.Research have showed that three NOS isoforms all play roles in heart disease mediated by β3-AR,but which NOS isoforms play a major role in myocardial depression in sepsis and the relationship with β3-AR is unclear.In this study,the object are LPS-induced septic shock in rats(in vivo,isoloted cells)and LPS-induced primary cultured cardiomyocytes,and we will observe the myocardial depression in time,detect β1,2,3-AR and NOS subtypes changes(protein,mRNA levels),detect β3-AR and NOS isoforms levels of primary cells(protein,mRNA levels)according the result of vivo,and study the mechanism of β3-AR in LPS-induced myocardial depression by using β3-AR agonist and NOS inhibitors.Materials and Methods1.Preparation of animal models⑴Rat model of septic shockIn this study,we use intravenous administration of endotoxin method to establish rat model of sepsis.Rats were anesthetized by 20% urethane solution 5ml/kg intraperitoneal injection,cutting left groin skin layers and blunting dissection of tissue and muscle to expose the left femoral artery and connect Biopac multi-conductive polygraph through the three-way head with pressure transducer(pre-discharge all the bubbles in the piping)for continuous monitoring of mean arterial pressure(MAP);to expose femoral vein for administration of LPS 15mg/kg(soluble 3ml/kg liquid),the pump speed is 0.1ml/min,regarded as septic shock when the basis of the value of MAP reduced by 25-30%.⑵Animal groups(1)Using septic shock time points to determine the experimental groups: random number table to Wistar rats were divided into LPS group and control group,10 animals in the control group received saline pumped into the femoral vein 3ml/kg,pump speed as before.Both groups of rats in vivo continuous monitoring of cardiac function for 6h.(2)Specimens were collected for experiments: the random number table Wistar rats were divided into six groups of 10 animals.Based on the test results of the analysis(1)experimental group were divided according to different time points after administration of LPS 2h,4h,6h group,the control group were setted up by the experimental group at each time point,given saline pumped into the femoral vein 3ml/kg,pump speed with the former.2.Specimen collection and handling of animalsExperiment(2): each group of rats were given to the respective time points after treatment abdominal aortic blood(3-4ml),then we cut the chest quickly,placed heart clipping in saline at 4 ℃ for residual blood to pump out,chosed specimens of left ventricle,-80 ℃ refrigerator for Real-timePCR and Western Blot test.Placement of arterial blood collected by centrifugation after 15 min,3000r/min centrifugal 10 min,the supernatant was loaded into EP tube,-80 ℃ refrigerator for ELISA testing.3.Acutely isolated rat cardiomyocytesSelected LPS 4h rats in the experimental group,the control group rats were given saline to the same point in time,with the former administration.Langendorff apparatus was used to retrograde aortic perfusion,and digestive constant perfusion was used to isolate single cardiomyocytes4.Primary culture of neonatal rat myocardial cellsSelected one day Wistar rats after the birth of,took the left ventricle muscle under sterile conditions,using a magnetic stirrer,trypsin and collagenase mixed digestion method to separate myocardial cells for primary culture,and given LPS incubation for calcium transient measurements and molecular biology.5.Experimental methods and testing indicators⑴Left ventricular cannulation and in vivo measurement of cardiac function.⑵ELISA was used to detect serum troponin I(cTn I)and creatine kinase MB(CK-MB).⑶Real-timePCR was used to detect mRNA expression of myocardial β1,2,3-AR and various subtypes NOS.⑷Western Blot was used to detect protein expression of myocardial β1,2,3-AR and various subtypes NOS.⑸Single myocardial contraction / relaxation function and calcium transient.⑹Measurement of multiple primary cultured cardiomyocytes intracellular calcium transients⑺Using Real-timePCR to detect mRNA expression of β1,2,3-AR and NOS2 in myocardial.⑻Using Western Blot to detect protein expression ofβ3-AR and NOS2 in myocardial cells.⑼Self-control of multi-cellular calcium transient of different drug groups.6.Statistical analysisNormally distributed data were expressed as mean ± standard deviation(X± SD),using t test for independent samples between the two groups,single-factor analysis of variance(ANOVA)was used to compare multiple groups,repeated measurements analysis of variance was used for continuous monitoring data.Using spss13.0 statistical software for data analysis,P <0.05 was considered statistically significant.Result1.MAP and left ventricular function changes of rats in septic shock in vivoThe blood pressure decline 25-30% compared to baseline values on 2h after the rats were given intravenous infusion of LPS.Dynamic monitoring of changes in MAP,there is a change in the model group compared with the control group,the overall trend is statistically significant(P <0.05),MAP values of model group 2h,4h,6h were statistically significant compared 0h value(P <0.05),while the control group MAP change over time was not statistically significant,which indicated that the septic shock model produced by this method is a stable model.The maximum rate of left ventricular systolic pressure rise(+ dp / dtmax)is statistically significant in the model group compared with the control group(P <0.05),model group 2h,4h,6h value were statistically significant compared to 0h baseline values(P <0.05),while the control group + dp / dtmax had no significant change over time.The maximum rate of left ventricular diastolic pressure drop(-dp/dtmax)change in the model group was statistically significant compared with the control group(P <0.05),model group 4h,6h value were statistically significant compared to the baseline 0h(P <0.05),while the control group-dp/dtmax had no significant change over time.2.Serum cTnI septic shock in rats and CK-MB changesThe serum of cTn I and CK-MB by dynamic monitoring in septic shock rat were not found statistically significant compared with the control group was(P> 0.05).No clear myocardial tissue necrosis or damage were found in this modle.3.β1,2,3-AR protein and mRNA expression in rat myocardium in septic shockβ3-AR protein level of myocardial tissue of rats with septic shock in early shock(model 2h)increased significantly compared to the control group(P <0.05),and significantly declined in late shock(model 6h)compared with the control group(P <0.05).The mRNA levels of myocardial β3-AR consistently changed.The protein and mRNA levels of β1,2-AR with the same point in time in the control group was not statistically significant.4.Various types of NOS protein and mRNA expression in rat myocardium in septic shockNOS2 protein level was not statistically significant compared with control group in rat myocardium in early shock,and the trend increased in 4h,6h after LPS treatment compared with the control group with the same time(P <0.05),and myocardial tissue NOS2 mRNA levels were higher at all time points(P <0.05).The change of NOS1 and NOS3 protein levels each time point were not statistically significant compared with the control group,NOS1 mRNA levels in LPS treated at 2h significantly increased(P <0.01),NOS3 mRNA levels at each time point were lower than control group(P <0.05),but the protein levesl of these two NOS isoforms changed inconsistently with mRNA level,which indicated that the two subtypes may not play a major role in this model.5.Single cardiomyocytes contraction / dilation of rats in septic shockSingle myocardial cells of LPS-treated for 4h rats were chosed to study,the contraction amplitude(peak shorting,PS),systolic / diastolic maximum velocity integral(+ d L / dt,-d L / dt)decreased compared to control group(P <0.01);(time to 90% PS,TPS90)and(time to 90% relengthening,TR90)prolonged compared to control group(P <0.01).6.Single cardiomyocytes calcium transient of rat septic shockThe resting calcium concentration(baseline FFI),the calcium concentration peak(peak FFI)and calcium transient amplitude(deta FFI)of single myocardial cell of LPS-treated 4h rats decreased compared to the control group(P <0.05);calcium returned to baseline at 90% of the level(TR90)and the decay time constant of calcium(tau)prolonged.(P <0.01).7.Calcium transients of LPS-induced primary cultured cardiomyocytesPrimary cultured neonatal rat cardiac cells with a final concentration of 1μg/ml LPS effect of 8,16,24 h,the state of 24 h group cell was poor,so it did not participate in the determination of calcium transients.Compared with normal control group,significant changes occurred in calcium transient in LPS16 h group,expressed as calcium transient amplitude(detaF/F0)decreased calcium concentration time course of recovery of 50%(TR50)extend and calcium concentration decay time constant(tau)increase(P <0.05),whereas LPS 8h group change was not statistically significant compared with the normal group.8.β3-AR protein and β1,2,3-AR mRNA changes of primary cultured cardiomyocytes by LPS induced for 16 hoursPrimary cultured myocardial cells were incubated for 16 h after LPS,and β3-AR protein and mRNA levels increased compared with the control group(P <0.05),while the β1,2-AR mRNA levels than those in the control group showed no significant change.9.NOS2 protein and mRNA changes of primary cultured cardiomyocytes by LPS induced for 16 hoursPrimary cultured myocardial cells were incubated for 16 h after LPS,and NOS2 protein and mRNA levels increased compared with the control group(P <0.05)10.Multicellular calcium transient of self-control results in different drug groupsLPS + BRL-37344 group and LPS + BRL-37344 + L-NAME group with normal calcium transients overall trend after the group has primary cultured cardiomyocytes 16 h incubation with LPS was statistically significant(P <0.05),LPS + BRL-37344 + CGP group and LPS + BRL-37344 + ICI group with normal calcium transients group overall trend was not statistically significant.Conclusion1.The septic shock rat model by LPS-induced is stable and cardiac function was prolonged and sustained downward over time.Evidence of myocardial injury does not appear.β3-AR may play a protective role in the whole process of septic shock.NOS2 played a major role in septic shock in the whole process than other two subtypes,and its relations with the β3-AR needs further study.2.Myocardial cells of septic shock rat still exists myocardial contractility / diastolic dysfunction and calcium dyshomeostasis other than neurohormonal.3.The calcium transients of primary cultured neonatal rat cardiomyocytes at a final concentration of 1μg/ml LPS-induced for 16 h reduced,which possiblely related to negative inotropic effect of β3-AR through NOS / NO pathway.