Effect Of Dobutamine On Intrinsic Myocardial Function And Myocardial Apoptosis In Septic Rats With Myocardial Dysfunction

Objective:Myocardial dysfunction is a common complication in septic patients,which is associated with increased mortality.But up till now,there is no specific effective therapy for this condition.In 2016,Surviving Sepsis Campaign Guidelines recommended that dobutamine,a?₁-adrenoceptor?AR?agonist,was the first-choice inotrope for septic patients with measured or suspected low cardiac output despite adequate fluid resuscitation and the use of vasopressors.However,we have recently demonstrated that?₁-AR antagonist inhibits myocardial apoptosis in endotoxemic mice,and there is no direct evidence to demonstrate if dobutamine treatment in the presence of intrinsic myocardial dysfunction improves myocardial function during the late phase of sepsis.The purpose of the present study was to determine the effect of dobutamine treatment in the presence of intrinsic myocardial dysfunction on myocardial function and apoptosis in septic rats exposed to cecal ligation and puncture?CLP?.Methods:The first part:the time pattern of sepsis-induced intrinsic myocardial dysfunction and other organ dysfunction in mild CLP rats.Male SD rats were randomly divided into sham group and CLP group.In order to establish a rat model of sepsis induced by mild CLP,the cecum was exposed,ligated at 1.2 cm length of the cecum and punctured.The survival rates of rats were recorded after sham operation or CLP.In separate experiments,at 6 h,9 h and 12 h after sham operation or CLP,the rat hearts were harvested and the intrinsic myocardial function was determined by a langendorff apparatus,the levels of serum aspartate aminotransferase?AST?and alanine aminotransferase?ALT?activities as well as blood urea nitrogen?BUN?were analyzed by the automatic biochemical analyzer7600,and the lung wet-dry weight?W/D?ratios were calculated.The second part:the time pattern of sepsis-induced intrinsic myocardial dysfunction and other organ dysfunction as well as changes in the inflammatory response and serum biomarker of myocardial injury in severe CLP rats.Male SD rats were randomly divided into sham group and CLP group.The cecum was exposed,ligated at 1.5 cm length of the cecum and punctured to establish a rat model of sepsis by severe CLP.The survival rates of rats were recorded after sham operation or CLP.In separate experiments,at 6 h,9 h and 12 h after sham operation or CLP,the rat hearts were harvested and the intrinsic myocardial systolic and diastolic function was determined by a langendorff system.Furthermore,the levels of serum tumor necrosis factor-??TNF-??,cardiac troponinI?cTn I?,N-terminal probrain natriuretic peptide?NT-proBNP?and heart-type fatty acid–binding protein?H-FABP?were measured by enzyme-linked immunosorbent assay?ELISA?,the mRNA expression of cardiac intercellular adhesion molecule-1?ICAM-1?,TNF-?and vascular cell adhesion molecule-1?VCAM-1?were measured by real time PCR,the levels of VCAM-1 protein were determined by western blotting assay.In addition,ALT activity,creatinine?Cr?and BUN concentration in the serum were analyzed by the automatic biochemical analyzer 7600,and the lung wet-dry weight?W/D?ratios were calculated.The third part:The effect of dobutamine treatment on survival,intrinsic myocardial function,serum and myocardial inflammatory cytokines,cTn I phosphorylation and myocardial apoptosis in severe CLP-induced septic rats with intrinsic myocardial dysfunction.Male SD rats were randomly divided into five groups,sham group,CLP group,CLP+2.5?g/kg/min DOB group,CLP+5.0?g/kg/min DOB group,CLP+10.0?g/kg/min DOB group.At 6 h after sham operation or CLP,DOB or normal saline was administered by the jugular vein.For survival assessment,rats were observed for10 d after the sham surgery and CLP operations.In separate experiments,male SD rats were randomly divided into six groups.sham group,CLP group,CLP+5.0?g/kg/min DOB group,CLP+10.0?g/kg/min DOB group,sham+5.0?g/kg/min DOB group and sham+10.0?g/kg/min DOB group.At 20 h after sham operation and CLP surgery,the hearts were isolated and mounted in a Langendorff system to measure intrinsic myocardial systolic and diastolic function.The levels of serum TNF-?,interleukin-1??IL-1??,interleukin-10?IL-10?and myocardial TNF-?and IL-1?were measured by ELISA.The cardiac caspase-3 activity was determined by a caspase-3 assay kit.In addition,the level of cleaved caspase-3 and the phosphorylation of cardiac troponin I?cTn I?protein was detected by Western blot,and myocardial apoptosis was determinedbyterminaldeoxynucleotidyltransferase-mediateddUTP nick-end-labeling?TUNEL?assay.Results:1.The survival rate of the mild CLP rats was 73.3%on day 10 after CLP induction.At 9 h and 12 h after CLP,the maximum rate of ascending and descent in left ventricular pressure?±dP/dt?of heart was decreased in CLP group compared with sham group?P<0.05?.At 12 h,serum ALT activity was increased in CLP group compared with sham group.However,there was no difference in serum BUN concentration and lung wet-dry weight?W/D?ratios at 6 h,9 h and 12 h after sham operation or CLP between CLP and sham group?P>0.05?.2.The survival rate of the severe CLP rats was 36.4%on day 10 after CLP induction.At 6 h,9 h,12 h after CLP,the maximum rate of positive and negative change in left ventricular pressure?±d P/dt?of heart was decreased in CLP group compared with sham group?P<0.05?.At 6 h,9 h and 12 h after CLP surgery,the serum TNF-?concentration as well as cardiac TNF-?,ICAM-1 and VCAM-1 mRNA and VCAM-1protein levels were significantly higher in CLP group than those in sham group.However,there was no significant difference in serum NT-proBNP,H-FABP,cTnI and Cr,BUN concentrations and lung wet-dry weight?W/D?ratios at 6 h,9 h and 12 h after sham operation or CLP between CLP and sham group?P>0.05?.At 9 h and 12 h after CLP,serum ALT activity was increased in CLP group compared with sham group?P<0.05?.3.At 6 h after sham operation or CLP,2.5?g/kg/min DOB,5.0?g/kg/min DOB,10.0?g/kg/min DOB or normal saline were administered by the jugular vein in severe CLP rats.The survival rate of rats in the sham group was 100%on day 10 after sham surgery.The survival rate of rats was 45.5%on day 10 after CLP 10 days in CLP group.On the day 10 after CLP,there was no difference in survival rate among CLP+2.5?g/kg/min DOB group?60%?,CLP+5.0?g/kg/min DOB group?31.6%?and CLP group?P>0.05?.However,the survival rate of rats in CLP+10.0?g/kg/min group was 75%,which was higher than that in CLP group?P<0.05?.In separate experiments,In severe CLP rats,the maximum rate of positive and negative change in left ventricular pressure?±dP/dt?was decreased at 20 h after CLP in CLP group compared with sham group?P<0.05?.There was no markedly difference in left ventricular±dP/dt between CLP+5.0?g/kg/min DOB group,CLP+10.0?g/kg/min DOB group and CLP group at 20 h after CLP?P>0.05?.There was also no significant difference in the levels of the serum and cardial TNF-?and IL-1?as well as cardiac caspase-3activity and cleaved caspase-3 protein and cTn I phosphorylation between CLP+5.0?g/kg/min DOB group,CLP+10.0?g/kg/min DOB group and CLP group at 20 h after CLP?P>0.05?.TUNEL staining demonstrated 5.0?g/kg/min and 10.0?g/kg/min DOB treatment did not reduce myocardial apoptosis in septic rats?P>0.05?.However,treatment with 10.0?g/kg/min DOB at 6 h after CLP significantly increased serum IL-10 concentration in septic rats?P<0.05?.Conclusion:1.In a rat model of sepsis induced by mild CLP,the intrinsic myocardial systolic and diastolic dysfunction was present at 9 h after CLP;In a rat model of sepsis induced by severe CLP,the intrinsic myocardial systolic and diastolic dysfunction occurred at 6 h after CLP.The intrinsic myocardial systolic and diastolic dysfunction was a common complication at the early stage during sepsis,which occurred earlier than hepatic dysfunction and occurrence of abnormal serum Cr and BUN concentrations and lung wet-dry weight?W/D?ratios.Serum biomarkers,such as cardiac troponin I?cTnI?,N-terminal pro-brain natriuretic peptide?NT-proBNP?and heart-type fatty acid–binding protein?H-FABP?,do not indicate the occurrence of intrinsic myocardial systolic and diastolic dysfunction at the early stage of sepsis.2.For septic rats induced by severe CLP,DOB treatment in the presence of intrinsic myocardial dysfunction?at 6 h after CLP?did not improve the myocardial function,and not affect myocardial inflammation and myocardial cell apoptosis in the late sepsis?at 20 h after CLP?.These results do not support the clinical use of DOB in the treatment of sepsis-induced cardiac dysfunction.3.For septic rats induced by severe CLP,treatment with 10.0?g/kg/min DOB after the occurrence of myocardial systolic and diastolic dysfunction significantly improves survival in septic rats,and its mechanism might be related to its promotion of anti-inflammatory cytokine IL-10 production.