The Research Of Rational That Mild Hypothermia Ameliorates Post-resuscitated Myocardial Dysfunction Caused By Epinephrine

Epinephrine has been the preferred vasopressor agent for cardiopulmonary resuscitation last 60 years.Its efficacy is due to its α-adrenergic vasopressor effect which increases coronary and cerebral perfusion during resuscitation.The potential adverse effects of epinephrine found recently,including ventricular dysrhythmias and post-resuscitation myocardial dysfunction,have been attributed primarily to its positive inotropic and chronotropic effects as a β1-adrenergic agonist.Mild hypothermia,also called therapeutic hypothermia,usually refers to that the core temperature covers from 32 ℃ ~35 ℃.Mild hypothermia exerts post-resuscitation neurologic protection in CPR animal model.Furthermore,it has been list in the AHA guideline regarding the treatment of cardiac arrest due to its post-resuscitation neurologic protection.Hypothermia is usually induced at the time when return of spontaneous circulation has been achieved in clinical treatment.But the data from animal experiment exhibit that the sooner when hypothermia induced after cardiac arrest,the better neurologic function achieve.Thus,the present study adopt the strategy that starting cooling at the beginning of CPR.Except for nervous system,the post-resuscitation protection role of other organ including heart for mild hypothermia has distracted more and more attention.Post-resuscitation myocardial dysfunction is to be with poor prognosises,it however could be alleviated by mild hypothermia.The present thesis firstly investigated the effect of epinephrine on post-resuscitation myocardial function and survival in CPR rat model given various resuscitation mode with either temperature or epinephrine as intervention factor and addressed that epinephrine aggravate post-resuscitation myocardial impairment which could be alleviated by epinephrine.The further molecular biology experiment was to explore the activity and expression of adrenoceptors in post-resuscitation myocardial cell membrane and showed that the role of mild hypothermia attenuating epinephrine inducing post-resuscitation myocardial dysfunction is associated with both inhibiting β1-adrenoceptor signaling and upregulating β1-adrenoceptor expression,but not with the activity and expression of α1-adrenoceptor.The third experiment inspired by above novel results proved that it was the activation of GRK2-β-arrestin2 signal pathway that accelerates the desensitization and recycling of post-resuscitation myocardium β1-AR when epinephrine was given with intra-arrest hypothermia resuscitation.Then,the reserve of active β1-AR was increased to keep the post-resuscitation myocardium response to endogenous catecholamine and protect the myocardial function.The present research innovatively explores the molecular rational of mild hypothermia attenuating post-resuscitation myocardial function impairment and will become the solid base for future research to find the medicine targeting to GRK2-β-arrestin2 signal pathway and substituting for hypothermia.This thesis is divided into 3 parts:PART I: The comparison of the effects of epinephrine on outcomes of normothermic and mild hypothermic cardiopulmonary resuscitationObjectives: To investigate the effects of epinephrine when administered during either normothermic or mild hypothermic cardiopulmonary resuscitation on postresuscitation myocardial function and survival in the electricity induced ventricular fibrillation rat model.Methods: 24 male SD rats were randomly assigned to one of four groups in the first part: normothermic placebo control(control+N);normathermic epinephrine(EPI+N);hypothermic placebo control(control+H);and hypothermic epinephrine(EPI+H).Ventricular fibrillation was induced electrically and untreated for 8 mins.Hypothermia was initiated coincident with the start of cardiopulmonary resuscitation and maintained at 33 ± 0.2°C for 4 hrs after resuscitation when the animals were euthanized for ventricular tissue harvest.Normothermia groups maintained core temperature at 37 ± 0.2°C throughout the study.Either placebo or epinephrine(20ug/kg)was administered 5 mins after the start of cardiopulmonary resuscitation and 3 mins before defibrillation.Post-resuscitation cardiac output,ejection fraction,and myocardial performance index were measured hourly for 4 hrs.Durations of survival observed for up to 3 months.Results: Except for 3 normotheric control rats,all animals were resuscitated.when epinephrine was administered during normothermic cardiopulmonary resuscitation,post-resuscitation myocardial function was severely impaired when compared with the normothermic control group.However,post-resuscitation myocardial function was significantly better in animals treated with epinephrine during hypothermic cardiopulmonary resuscitation when compared with hypothermic controls.This was associated with significantly fewer post-resuscitation ventricular arrhythmias and longer duration of survival.Conclusions: Epinephrine,when administered during normothermic cardiopulmonary resuscitation,significantly increases the severity of postresuscitation myocardial dysfunction and decreases the duration of survival.These detrimental effects of epinephrine,however,no longer exist when it is administered during therapeutic hypothermic cardiopulmonary resuscitation.PART II The role of mild therapeutic hypothermia attenuating epinephrine inducing post-resuscitation myocardial dysfunction is associated with both inhibiting β1-adrenoceptor signaling and upregulating β1-adrenoceptor expressionObjective:Epinephrine administered during cardiopulmonary resuscitation is associated with severe post-resuscitation myocardial dysfunction.We have previously demonstrated therapeutic hypothermia reduced the severity of post-resuscitation myocardial dysfunction caused by epinephrine,however,the mechanism of this myocardial protective effect remains unclear.The present study is to explore the association of adrenoceptors expression and signal transduction with mild hypothermia induced myocardial function protection.Methods: Rats weighing between 450-550 g were randomized into four groups: 1)normothermic placebo;2)normothermic epinephrine;3)hypothermic placebo;and 4)hypothermic epinephrine.Ventricular fibrillation was induced and untreated for 8 mins.Hypothermia was initiated coincident with the start of CPR and maintained at 33 ± 0.2°C for 4 hrs.Either placebo or epinephrine was administered 5 mins after the start of CPR and 3 mins before defibrillation Post-resuscitation ejection fraction was measured hourly for 4 hrs.Hearts were then harvested for further analysis.Results: Epinephrine increased coronary perfusion pressure during CPR(26.5±6.23 mm Hg vs 21.14±1.95 mm Hg P<0.05).Post-resuscitation myocardial function was impaired in the normothermic epinephrine group compared with the other three groups.The concentration of myocardial c AMP was doubled in the normothermic epinephrine group(655.06±447.63 umol/L)compared to the hypothermic epinephrine group(302.51±97.98 umol/L P<0.05).Myocardial β1-adrenoceptor expression was elevated in the animals treated with hypothermia regardless of epinephrine.Conclusion: Epinephrine,when administered during normothermic cardiopulmonary resuscitation,increased the severity of post resuscitation myocardial dysfunction.This adverse effect was inhibited by intra-arrest hypothermia.The potential mechanism may be associated with reduced activity with up-regulation of β1-receptors after hypothermia.Parts III Activation of GRK2-β-arrestin2 signal pathway plays a key role in the mild hypothermia ameliorating epinephrine-linked post-resuscitation myocardial dysfunctionObjectives: desensitization,degradation or recycling of adrenoceptors on the myocardium membrane is significant in the receptors regulation.There are two ways including homologous and heterologous desensitization.The first part of the study is to explore the expression of the key proteins or enzymes determining above two ways and the relationship between activation of receptors desensitization and post-resuscitation myocardial function protection by mild hypothermia.The second part is to investigate whether activation of GRK2-β-arrestin2 signal pathway is the key point regarding mild hypothermia ameliorating epinephrine-linked post-resuscitation myocardial dysfunction through GRK2.inhibition.Method: 12 male SD rats were randomly assigned to one of two groups in the first part: normathermic epinephrine(EPI+N)and hypothermic epinephrine(EPI+H).Ventricular fibrillation was induced electrically and untreated for 8 mins.Hypothermia was initiated coincident with the start of cardiopulmonary resuscitation and maintained at 33 ± 0.2°C for 4 hrs after resuscitation when the animals were euthanized for ventricular tissue harvest.Normothermia groups maintained core temperature at 37 ± 0.2°C throughout the study.Either placebo or epinephrine was administered 5 mins after the start of cardiopulmonary resuscitation and 3 mins before defibrillation.Post-resuscitation cardiac output,ejection fraction,and myocardial performance index were measured hourly for 4 hrs.Animals were sacrificed at 4 hrs after return of spontaneous circulation and the left ventricular myocardium specimen was obtained to further examination.The expression of total and phosphorylated β1-AR,GRK2,β-arrestin2 and PKA were tested through western blotting,β-arrestin2 m RNA though real-time q PCR.The other 12 male SD rats were randomly assigned to one of two groups in the first part: Paroxetine and control.Rats in the Paroxetine group were pre-feed with paroxetine(10mg/kg/day)for 2 weeks.All the animals were induced VF and then CPR with intra-arrest hypothermia.The data including myocardial function,neurologic function score and survival were recorded.Results: the first part: the post-resuscitation myocardial function in EPI+H group was better than it in EPI+N group.Total and phosphorylated β1-AR expression on the myocardium membrane in EPI+H group had a significant increment compared to EPI+N group.Further,both GRK2 and β-arrestin2 expression significant elevated,while PKA expression significant decreased in EPI+H group.β-arrestin2 m RNA in myocardium also increased in EPI+H group significantly.The second part: More severe impaired post-resuscitation myocardial function,lower neurologic function score and shorter duration of survival were exhibited in the animals given paroxetine.Conclusion: Activation of c AMP-PKA signal pathway in myocardium is associated with myocardial function impairment in the animals given normothermic epinephrine resuscitation.The rationale could be that c AMP-PKA signal pathway activation accelerates β1-AR desensitization and degradation which result in decreased active β1-AR on myocardium membrane.Activation of GRK2-β-arrestin2 signal pathway plays a key role in the mild hypothermia ameliorating epinephrine-linked post-resuscitation myocardial dysfunction.The possible mechanism is that activation of GRK2-β-arrestin2 signal pathway accelerates β1-AR desensitization and recycling which result in increased active β1-AR on myocardium membrane.