Experimental And Clinical Study On Cardioprotection Of Preemptive Thyroid Hormone Administration Against Ischemia-reperfusion Injury

After the patients undergoing open heart surgery with cardiopulmonary bypass(CPB) experience global ischemia/reperfusion(I/R) injury during surgery,serum level of T₃ decreases significantly.This problem is especially common in pediatric patients.It has been demonstrated that all pediatric patients undergoing open heart surgery with CPB present the significant decreased serum levels of T₃ and T₄ after surgery,which is named as typeâ…¡Euthyroid sick syndrome(ESS) and may cause undesirable impact on the children's prognosis.The available main therapeutic measure aimed at the postoperative ESS is that when decreased serum levels of thyroid hormones are detected during postoperative period, supplemental administration of exogenous thyroid hormones restores the serum levels of thyroid hormones to normal limits.However,postoperative supplemental administration of exogenous thyroid hormones can not ensure to achieve consistent and adequate therapeutic effect for ESS.Also,it may further interfere with the disturbed inner environment after open heart surgery with CPB,which is adverse to recuperate the patients' health.When serum levels of thyroid hormones are restored to normal limits by the postoperative supplemental administration of exogenous thyroid hormones,moreover,the beneficial effects of ESS may also be abolished.It has been demonstrated that thyroid hormones can produce the definite cardioprotecton.In view of possible disadvantages of the postoperative supplemental administration of exogenous thyroid hormones,we consider that in pediatric patients undergoing the open heart surgery with CPB,preemptive administration of exogenous thyroid hormones before surgery may not only protect the myocardium against I/R injury during surgery by increasing the expression of myosin heavy chainα(MHCα) and heat shock protein 70(HSP70) in myocardial tissues,but also can avoid the adverse effects by the postoperative supplemental administration of exogenous thyroid hormones.However, there has been no published data on protection of myocardial I/R injury during surgery and prevention of postoperative ESS by administrating exogenous thyroid hormones before open heart surgery with CPB in pediatric patients. Therefore,we designed this study including an animal experiment and a clinical trial. Our purposes were to determine whether preemptive administration of exogenous thyroid hormones could protect the myocardium against I/R injury and to explore the inherent mechanisms of the cardioprotecton by preemptive administration of thyroid hormones.By this study,we hoped to provide some useful rationales for further use of this simple cardioprotective measure in clinical practice.The animal experiment of this study was divided into three subunits.Animal ExperimentPart 1.Protective effects of preemptive administration of different-dose levothyroxine-sodium against myocardial ischemia-reperfusion injuryAfter weighing,48 healthy female immature(aged 35 days) Wistar rats were randomly allocated into the six groups(8 rats in each group):blank control group(group BC),control group(group BC),10μg group(group 10μg),20μg group(group 20μg),40μg group(group 40μg) and 80μg group(group 80μg).The rats in groups BC and C were fed with normal food for 7 days before experiment.Except for feeding with normal food for 7 days before experiment,the rats in groups 10μg,20μg,40μg and 80μg were also pre-treated with levothyroxine-sodium 10μg/100g weight,20μg/100g weight,40μg/100g weight 80μg/100g weight,respectively,through a gastric tube every day.On the eighth day, all rats were again weighed and then underwent peripheral venous puncture to take blood sample for detection of serum levels of thyroid hormones.The hearts were harvested from the anesthetized rats,and appended to a Langendorff apparatus for isolated heart perfusion model.The hearts in group BC were perfused with KH fluid for 80 min without zero-perfusion,whereas the hearts in other groups experienced in order a 30-min balance period,a 20-min normothermic zero-perfusion and a 30-min reperfusion period.The hemodynamic variables including heart rate,systolic blood pressure(SBP),and±dp/dt_max were continuously monitored.The coronary perfusion flow was recorded at 10 min during balance period and at 10 min during reperfusion period,respectively.At 15 min during reperfusion period,the coronary perfusion fluid was collected for detection of cardiac enzyme-myocardial-bound creatine kinase(CK-MB).At the end of perfusion,the ventricular muscle samples were taken to detect expression of HSP70 by Western-blotting, and expression of both MHC mRNA and thyroid hormone receptor(TR) mRNA by quantitative RT-PCR.The results showed that compared with groups BC and C,rats' weight on the experiment day was significantly lower in groups 20μg,40μg and 80μg.Also,rats' weight on the experiment day was significantly lower in groups 40μg and 80μg than in group 10μg.The serum levels of thyroid hormones were significantly higher in groups 20μg, 40μg and 80μg than in groups BC and C.The serum levels of thyroid hormones in groups 10μg,20μg,40μg and 80μg increased with the dosage of the levothyroxine-sodium,and the serum levels of thyroid hormones were significantly higher in group 80μg than in groups 10μg,20μg and 40μg.As compared with groups BC and C,baselines of both heart rate and dp/dt_max were significantly higher in groups 10μg,20μg,40μg and 80μg.Baselines of SBP and -dp/dt_max were significantly higher in group 80μg than in groups 10μg and 20μg.As compared with group BC,recovery rates of heart rate during reperfusion period significantly decreased in groups 40μg and 80μg,and recovery rates of both SBP and±dp/dt_max significantly decreased in groups 10μg,20μg,40μg and 80μg.As compared with group C,recovery rates of both heart rate and dp/dt_max during reperfusion period significantly increased in groups 10μg,20μg,40μg and 80μg,recovery rate of SBP during reperfusion period significantly increased at 20 min and 30 min in group 10μg and at 30 min in group 20μg,recovery rate of -dp/dt_max during reperfusion period significantly increased at all observed time points in groups 10μg,20μg and 40μg,and at 30 min in group 80μg.Comparisons among all four experiment groups,recovery rate of SBP at 30 min during reperfusion period was significantly lower in group 80μg than in group 10μg, and recovery rate of±dp/dt_max at 30 min during reperfusion period was significantly lower in groups 40μg and 80μg than in groups 10μg and 20μg.As compared with groups BC and C,coronary perfusion flows in balance period and reperfusion period significantly increased in groups 10μg,20μg,40μg and 80μg.The activity level of CK-MB in the coronary perfusion fluid during reperfusion period was significantly higher in groups 10μg,20μg,40μg and 80μg than in group BC,but was significantly lower in groups 10μg,20μg and 40μg than in group C.Comparisons among all four experiment groups,the activity level of CK-MB in the coronary perfusion fluid during reperfusion period increased with the dosage of levothyroxine-sodium,and was significantly higher in group 80μg than in groups 10μg,20μg and 40μg.The ventricular myocardial expression of HSP70 was stronger in four experiment groups than in groups BC and C,and significantly increased in groups 10μg and 20μg compared with groups 40μg and 80μg.As compared with groups BC and C,the ventricular myocardial expression of MHCαmRNA significantly increased in four experimental groups,and the ventricular myocardial expression of TRα1 mRNA significantly increased in groups 20μg,40μg and 80μg. However,there were no significant differences in the ventricular myocardial expression of MHCαmRNA and TRα1 mRNA among four experiment groups.Part 2.The influences of fentanyl and sufentanil on cardioprotection of preemptive levothyroxine-sodium administrationFifty-six healthy,female immature(aged 35 days) Wistar rats were randomly allocated into the seven groups(8 rats in each group):blank control group(group BC), control group(group C),levothyroxine-sodium 10μg group(group 10μg),fentanyl group (group F),sufentanil group(group S),combined fentanyl and levothyroxine-sodium(group F+L) and combined sufentanil and levothyroxine-sodium(group S+L).The rats in groups BC,C,F and S were fed with normal food for 7 days before experiment.Except for feeding with normal food for 7 days before experiment,the rats in groups F+L,S+L and 10μg were also pre-treated with levothyroxine sodium 10μg/100g weight,through a gastric tube every day.On the eighth day,the hearts were harvested from the anesthetized rats,and appended to a Langendorff apparatus for isolated heart perfusion model.The hearts in group BC were perfused with KH fluid for 80 min without zero-perfusion, whereas the hearts in other groups experienced in order a 30-min balance period,a 20-min normothermic zero-perfusion and a 30-min reperfusion period.The hearts were perfused with KH fluid containing fentanyl 30μg/L in the F and F+L groups,with KH fluid containing sufentanil 3μg/L in the S and S+L groups,and with KH fluid in other groups, respectively.The hemodynamic variables including heart rate,systolic blood pressure (SBP),and±dp/dt_max were continuously monitored.The coronary perfusion flow was recorded at 10 min during balance period and at 10 min during reperfusion period, respectively.At 15 min during reperfusion period,the coronary perfusion fluid was collected for detection of CK-MB.At the end of perfusion,the ventricular muscle samples were taken to detect expression of HSP70 by Western-blotting,and expression of both MHC mRNA and thyroid hormone receptor(TR) mRNA by quantitative RT-PCR.The results showed no significant differences in baselines of both heart rate and SBP among seven groups.Baselines of±dp/dt_max were significantly higher in groups 10μg,F+L and S+L than in groups BC,C,F and S. As compared with group BC,all hemodynamic variables during reperfusion period significantly decreased in groups F and S.As compared with group C,all hemodynamic variables during reperfusion period significantly increased in groups 10μg,F+L and S+L. All hemodynamic variables during reperfusion period were significantly higher in groups 10μg,F+L and S+L than in groups F and S.However,there were no significant differences in all hemodynamic variables during reperfusion period among groups 10μg,F+L and S+L.The coronary perfusion flows in balance period and reperfusion period significantly increased in groups 10μg,F+L and S+L compared with groups C,F and S.However,there were no significant differences in the coronary perfusion flows in balance period and reperfusion period among groups 10μg,F+L and S+L.The activity level of CK-MB in the coronary perfusion fluid during reperfusion period was significantly higher in groups 10μg,F+L and S+L than in group BC,but lower in groups 10μg,F+L and S+L than in groups C,F and S.The activity level of CK-MB in the coronary perfusion fluid during reperfusion period was not significant different among groups 10μg,F+L and S+L.The ventricular myocardial expression of HSP70 significantly increased in groups 10μg,F+L and S+L compared with groups C,F and S.However,there were no obvious differences in the ventricular myocardial expression of HSP70 among groups 10μg,F+L and S+L.The ventricular myocardial expression of MHCαmRNA significantly increased in groups 10μg,F+L and S+L compared with groups C,F and S.However,the ventricular myocardial expression of MHCαmRNA was not significant different among groups 10μg, F+L and S+L.Also,there were no significant differences in the ventricular myocardial expression of TRα1 mRNA among all seven groups.Part 3.The relationship between mitochondrial K_ATP and cardioprotection of preemptive levothyroxine-sodium administrationThirty-two healthy,female immature(aged 35 days) Wistar rats were randomly allocated into the four groups(8 rats in each group):blank control group(group BC), control group(group C),levothyroxine-sodium 10μg group(group 10μg) and combined glibenclimide and levothyroxine-sodium group(group G+L).The rats in groups BC and C were fed with normal food for 7 days before experiment.Except for feeding with normal food for 7 days before experiment,the rats in groups 10μg and G+L were also pre-treated with levothyroxine-sodium 10μg/100g weight,through a gastric tube every day.On the eighth day,the hearts were harvested from the anesthetized rats,and appended to a Langendorff apparatus for isolated heart perfusion model.The hearts in group BC were perfused with KH fluid for 80 min without zero-perfusion,whereas the hearts in other groups experienced in order a 30-min balance period,a 20-min normothermic zero-perfusion and a 30-min reperfusion period.The hearts were perfused with KH fluid containing glibenclimide 30μmol/L in group G+L,and with KH fluid in other groups, respectively.The hemodynamic variables including heart rate,systolic blood pressure,and±dp/dt_max were continuously monitored.The coronary perfusion flow was recorded at 10 min during the balance period and at 10 min during reperfusion period,respectively.At 15 min during reperfusion period,the coronary perfusion fluid was collected for detection of CK-MB.At the end of perfusion,the ventricular muscle samples were taken to detect expression of HSP70 by Western-blotting,and expression of both MHC mRNA and thyroid hormone receptor(TR) mRNA by quantitative RT-PCR.The results showed no significant differences in baselines of both heart rate and SBP among four groups.However,baselines of±dp/dt_max significantly increased in groups 10μg and G+L compared with groups BC and C.As compared with group C,all hemodynamic variables during reperfusion period significantly increased in groups 10μg and G+L.However,all hemodynamic variables during reperfusion period were not significant different between groups 10μg and G+L.The coronary perfusion flows in balance period and reperfusion period significantly increased in groups 10μg and G+L compared with group C.Also,the coronary perfusion flow in balance period significantly increased in groups 10μg and G+L compared with group BC.However,there were no significant differences in the coronary perfusion flows in balance period and reperfusion period between groups 10μg and G+L.The activity level of CK-MB in the coronary perfusion fluid during reperfusion period was significantly lower in groups 10μg and G+L than in group C,but significantly higher in groups 10μg and G+L than in group BC.There were no significant differences in the activity level of CK-MB in the coronary perfusion fluid during reperfusion period between groups 10μg and G+L.The ventricular myocardial expression of HSP70 significantly increased in groups 10μg and G+L than in groups BC and C.However,there was no significant difference in the ventricular myocardial expression of HSP70 between groups 10μg and G+L.The ventricular myocardial expression of MHCαmRNA significantly increased in groups 10μg and G+L compared with groups BC and C.However,there was no significant difference in the ventricular myocardial expression of MHCαmRNA between groups 10μg and G+L.Also,the ventricular myocardial expression of TRα1 mRNA was not significant different among all four groups.Based on the results from all three animal experiments,the following conclusions could be drawn.1.Preemptive administration of small-dose levothyroxine-sodium can protect myocardium against I/R injury,whereas preemptive administration of larger dose levothyroxine-sodium can only produce an attenuated cardioprotection effect and might result in occurrence of hyperthyroidism.2.Of four dosages of levothyroxine-sodium used in this experiment,a dosage of 10μg/100g weight achieves the best cardioprotection effect without occurrence of hyperthyroidism.3.The cardioprotective effect of preemptive levothyroxine-sodium administration is probably contributed to the increased myocardial expression of HSP70 and MHCαmRNA.4.Both fentanyl and sufentanil do not influence the cardioprotective effect of preemptive levothyroxine-sodium administration.5.Mitochondrial ATP sensitive potassium channel is not probably involved in the mechanism of the cardioprotection by preemptive levothyroxine-sodium administration.Clinical trialThe cardioprotective effect of small-dose thyroid hormone tablet administration before open heart surgery with cardiopulmonary bypass in childrenForty children aged 3-12 year,scheduled for elective simple congenital heart diseases (atrial septal defect,ventricular septal defect,patent foramen ovale and patent ductus arteriosus) surgery with cardiopulmonary bypass(CPB) were included in this clinical trial. They were randomly allocated equally into two groups:control group(n=20) and trial group(n=20).The children in the trial group took thyroid hormone tablet 0.4 mg/kg weight every day for four days before surgery,while the children in the control group were not given this tablet.In perioperative period,all children were treated according to the standing guidelines of the hospital.The serum levels of thyroid hormones were detected at following observed points:administration day,before anesthesia,and 1st,2nd and 4th day after surgery.The hemodynamic parameters were recorded at following observed points: administration day,immediately after surgery,and 1st and 2nd day after surgery.Also, duration of intubation and ICU stay,and the application of inotropic drugs in the ICU were noted.During surgery,right atria samples were taken from six children randomly chosen in each group to detect the myocardial expression of HSP70 by Western-blotting and the myocardial expression of TR mRNA and MHC mRNA by RT-PCR.At 24 hour after surgery,the blood samples were also taken to quantitatively assess serum activity of CK-MB and to qualitatively assay the positive ratio of TnI by AXSYM automatic immunoassay systems.The results showed that compared with the baselines on the administration day,in control group,the serum levels of both T₃ and FT₃ on the 1st,2nd and 4th day after surgery, and the serum levels of TSH,T₄ and FT₄ on the 1st day after surgery significantly decreased.As compared with the baselines on the administration day,in trial group,the serum levels of T₃ and FT₃ on the 1st,2nd and 4th day after surgery,and the level serum TSH on the 1st day after surgery significantly decreased.The serum levels of T₃,FT₃ and T₄ on the 1st and 2nd day after surgery,the serum level of FT₄ on the 1st day after surgery were significantly higher in the trial group than in control group.The application rate of inotropic drugs in ICU,the serum activity of CK-MB,the positive ratio of TnI and the atrial myocardial expression of MHCβmRNA significantly decreased,and the atrial myocardial expression of HSP70 and MHCαmRNA significantly increased in the trial group compared with control group.Based on the results of this clinical trial,the following conclusions could be drawn.1.In children undergoing simple open heart surgery with CPB,Typeâ…¡ESS may occur after surgery. 2.A short-term administration of small-dose thyroid hormone tablet before surgery in children undergoing open heart surgery with CPB can reduce the severity of decreased serum levels of thyroid hormones after surgery and prevent occurrence of postoperative typeâ…¡ESS.However,typeâ… ESS may still occur after surgery in the children.3.A short-term administration of small-dose thyroid hormone tablet before surgery in children undergoing open heart surgery with CPB can produce definite cardioprotecton. Also,the cardioprotective effects by administration of small-dose thyroid hormone tablet before surgery may be contributed to the increased myocardial expression of HSP70 and MHCαmRNA.