The Effect And Mechanism Of Propofol On Systolic Function Of Normal And Ischemia-reperfusion Heart

BackgroudThe circulation function of patients will change with the difference dosage and types of anesthetic in the clinical operation process.Inhibition of myocardial contraction may lead to severe hypotension and even may decrease heart and brain organ perfusion rapidly and cause organ dysfunction after operation.The clinical anesthesia doctor has recognized that we should choose the proper anesthetics for the general anesthesia patients with multiple diseases(such as hypertension,coronary heart disease,diabetes)to suppress organ damage.During the process of clinical anesthesia,the inhibition effect of anesthetics on the circulatory system is mainly due to the inhibition of cardiac contractility.CAMP dependentprotein kinase(PKA)can activate receptors on the sarcoplasmic reticulum of cardiac myocytes,and plays an important regulatory role in the process of myocardial contraction.Cardiomyocyte agonist activates adenylate cyclase(AC)through excitatory G protein(Gs),which causes the increase of cAMP level in the myocardium,and cAMP reactivates PKA.The function of PKA activation on myocardial contractile regulation is mainly phosphorylation of PLN and RyR,thereby increasing the intracellular calcium concentration and eventually leading to myocardial contraction.Propofol,a commonly used intravenous anesthetic,is characterized by its rapid sedation and hypnotic effect.Patients can quickly wake up after stopping transfusion since its rapid metabolism.Clinical reports and basic experiments have shown that propofol has the inhibitory effect of circulatory function,partly suppressing myocardial contractile function and reducing cardiac output.This study used Langendorff isolated heart perfusion technique,excluding the impact of other organs to the heart,monitoring the effects of different concentrations of propofol on cardiac systolic function,expression and phosphorylation of PKA and its downstream PLN,RyR protein.The objective of study was analysis weather propofol could reduce myocardial contractility by inhibition calcium ion pathway PKA regulated.Propofol,a commonly used analgesic and anxiolytic drug,is used in clinical anesthesia and intensive care.Animal experiments show that propofol pretreatment can significantly reduce the incidence of ventricular arrhythmia and infarct size induced by ischemia-reperfusion.In the world,ischemic heart disease caused by complete or incomplete coronary occlusion is one of the most important factors of death.Reperfusion immediately after ischemia can prevent cardiac arrest,but myocardial ischemia reperfusion injury(MIRI)will be more serious injuy.Ischemia reperfusion injury is more common in clinical anesthesia.Multiple coronary and cardiac surgery(such as coronary artery thrombolysis,coronary angioplasty,percutaneous coronary intervention)with aortic clamping,myocardial reperfusion injury will occur myocardial ischemia,affecting the prognosis of patients.MIRI is characterized by local cell oxidative breakdown,apoptosis and local inflammatory response induced by tissue dysfunction.During reperfusion,a variety of inflammatory factors such as IL-1,IL-6 and TNF-alpha are released,resulting in excessive local inflammatory response.CaMKII is excessively activated during this process,causing disturbance of PLN and RyR2 activity,which may lead to calcium overload and then cause myocardial damage.In this study,isolated heart ischemia reperfusion model and myocardial ischemia reperfusion model in vitro were used to monitor cardiac function after propofol or KN93 intervention,and to observe the effect of propofol on CaMKII regulation in ischemia-reperfusion injury.Part One Propofol reduced myocardial contraction of vertebrates partly by mediating the cyclic AMP-dependent protein kinase phosphorylation pathwayObjectiveThe inhibition mechanism of propofol on circulation system is not clear in the process of clinical anesthesia.The previous study showed that propofol has a direct inhibitory effect on the heart.This study used Langendorff isolated heart perfusion technique to exclude the impact of other body organs to the heart,monitoring the effects of different concentrations of propofol on cardiac systolic function,expression and phosphorylation state of PKA and its regulation downstream protein PLN,RyR.The objective of the study is to explore whether propofol reduced the myocardial systolic function and prove propofol could reduce myocardial contraction by mediating PKA activity without beta agonists.MethodsThis research was validated adopting the method of isolated organ,cell and molecule level experiments:the isolated rat hearts were suspended on Langendorff perfusion system and balloon filled with water was inserted into the left ventricle.Index of left ventricular contraction was monitored and perfusion K-H liquid with propofol solution concentrations were 10 20,30,40,50,100 N.M.Rat hearts were perfused by K-H liquid with 50 ?M propofol or PKA inhibitor(H89)and then collected.The heart tissues were extracted mRNA and related proteins and partially submerged in 4%paraformaldehyde to make frozen section.The content of cAMP and the activity of PKA in tissues were measured using ELISA method.PKA mRNA expression was determined by RT-PCR and qPCR and PKA?P-PLN/PLN and P-RyR2/RyR2 protein expression were measured by western bloting.Phosphorylated PKA protein and PKA activity in myocardial tissue was detected by immunofluorescence assay and phosphorylated PKA protein assay kit without radioactive reagent.Results1.The effect of different concentrations of propofol on myocardial contractilityCompared with the normal heart,the left ventricular systolic amplitude of the isolated heart was significantly decreased after infusion of propofol,and showed a dose-dependent inhibition effect.2.The effect of propofol(50 ?M)and H89 on cardiac functionWe investigated the dose-dependent effect of propofol to choose optimum concentration that would match clinical concentration.Panel demonstrates that propofol at a dose of 50 ?M showed a significant decline in cardiac contractive function which showed cardiac contraction amplitude declined to 55%of control.With the parameters of myocardial function containing left ventricular developed pressure(LVDP),maximum rate of pressure development in the left ventricle(dP/dt max),minimum rate of pressure development in the left ventricle(dP/dt min),heart rate(HR)recorded,the inhibition of cardiac contraction LVDP by propofol was decreased in the presence of H89.After washing,the amplitude recovered and then propofol decreased the cardiac amplitude.Propofol showed similar effect on dP/dt max and dP/dt min,while HR was significantly decreased only by propofol.3.Effect of propofol and H89 on cAMP production and PKA activation.We found that there was no significant difference between the dose groups in relation to the level of cAMP in the hearts.However,activity of PKA treated with propofol(50-300 mM)decreased significantly compared with lower doses of propofol.4.Effect of propofol(50 ?M)on PKA,PLN,RyR2 mRNA production.Following treatment with H89 and propofol respectively for 30 min,PKA,PLN and RyR2 mRNA levels were not altered.Similarly,propofol did not change the total PKA and downstream protein production.However,50?M propofol treatment reduced PKA activity significantly.5.Effect of propofol on PKA phosphorylation verified by western blotting.50 ?M propofol,similar to H89,reduced the expression of P-PKA.What's more,propofol decreased the phosphorylation levels of PLN and RyR2.However,propofol did not alter the production of total protein levels of PKA,PLN and RyR2.The levels of mRNA of PKA and PLN also showed no significant difference.6.Evidence about the direct effect of propofol on PKA activity usingimmunofluorescence and non-radioactive kitTo verify the direct effect of propofol on PKA activity,total PKA and the phosphorylation of PKA were detected by immunofluorescence kit.The results showed that propofol and H89 reduced the activation of PKA but not altered the total PKA levels.And similarly,propofol decreased the phosphorylation levels of PKA.Conclusions1.Propofol inhibited cardiac contraction in vitro in a dose-dependent manner,but did not affect cAMP content.2.Propofol inhibited cardiac systolic function by inhibiting PKA regulating PLN,RyR2 receptor phosphorylation.3.Without beta adrenergic receptor agonist,propofol reduced the activity of PKA to inhibit myocardial contraction.Part Two Effect of propofol regulating CaMKII on myocardial ischemia reperfusion injuryObjectiveThe features of MIRI are local cell apoptosis and oxidative burst,causing inflammation tissue dysfunction during reperfusion,releasing a variety of inflammatory factors.CaMKII is excessively activated during this process,causing disturbance of PLN and RyR2 activity,which may lead to calcium overload and then cause myocardial damage.In this study,isolated heart ischemia reperfusion model and myocardial ischemia reperfusion model in vitro were used to monitor cardiac function after propofol or KN93 intervention,and to observe the effect of propofol on CaMKII regulation in ischemia-reperfusion injury.MethodsIsolated heart ischemia reperfusion model of rat heart was used Langendorff perfusion suspension system and balloon filled with water was inserted into the left ventricle.Index of left ventricular contraction was monitored.After stabling the heart condition 20min,stop perfusion fluid 30min and reperfuse liquid 1h.CaMKII inhibitor KN93 or propofol were added into the perfusate respectively to observe cadiac function indexes.Collect the coronary outflow fluid during stable phase,ischemia-reperfusion 10min and 60min,and measure LDH,cTnI by ELISA.The heart tissues were collected in different periods.Myocardial apoptosis was detected by western blot.The levels of IL-1,IL-6 and TNF-alpha mRNA were detected by qPCR.The phosphorylation expression levels of CaMKII and PLN and RyR2 protein were detected by western blot.Primary cultured myocardial cells,myocardial cells produced in vitro ischemia reperfusion injury model,respectively adding KN93 or propofol intervention,measuring the phosphorylation expression levels of CaMKII,PLN and RyR2 using Western blotting.Results1.Propofol can stabilize the cardiac function after ischemia-reperfusionCompared with IR group,propofol and KN93 promote recovery of systolic function after ischemia reperfusion injury in vitro.Propofol and KN93 treatment group,the recovery of systolic function and decreased the frequency of rapid ventricular tachycardia and ventricular fibrillation.2.The effect of propofol on the levels of CaMKII,PLN and RyR2 phosphorylation after myocardial ischemia reperfusion injury.Propofol and KN93 significantly decreased the phosphorylation level of CaMKII,PLN Thrl7 and RyR2 S2814 after myocardial ischemia and reperfusion,but had no effect on the total protein level.3.The effect of propofol on necrosis marker of coronary outflow fluid and apoptosis regulating protein of cardiac tissuePropofol and KN93 can reduce the content of LDH and cTnI in coronary flow after myocardial ischemia and reperfusion.Propofol and KN93 can reduce the protein expression of Bax,enhance the expression of Bcl-2 and inhibit the apoptosis of myocardial cells after ischemia-reperfusion.4.The effect of propofol on the inflammatory factors in the cardiac tissue after ischemia-reperfusion.Propofol and KN93 can significantly reduce the contents of IL-1,IL-6 and TNF-alpha in the heart tissue after ischemia reperfusion.It shows that the effect of propofol and KN93 on the inflammatory factors in the heart tissue may be related to CaMKII.5.Effect of propofol on the phosphorylation levels of CaMKII,PLN and RyR2 in myocardial cells after ischemia-reperfusion.Propofol and KN93 can significantly reduce the level of phosphorylation of CaMKII,PLN and RyR2 protein after myocardial ischemia and reperfusion.Conclusions1.Propofol can stabilize the cardiac function after ischemia-reperfusion and reduce the incidence of VT and VF.2.Propofol and KN93 can reduce the phosphorylation levels of CaMKII,PLN and RyR2 after ischemia-reperfusion injury.3.Propofol and KN93 can alleviate the injury of myocardial cells after ischemia and reperfusion,reduce the production of inflammatory factors and myocardial necrosis markers,and reduce the apoptosis of cardiomyocytes.