Protective Effects And Mechanism Of Amiodarone In Rats With Stress Cardiomyopathy

Stress cardiomyopathy(SC),also known as Takotsubo cardiomyopathy,is a syndrome of temporary,marked regional left ventricular systolic disturbances occurring after mental or physical stress.Patients with SC typically present with chest pain and dyspnea and often have ST-segment elevation on the ECG;therefore,up to 1-2% of patients who are suspected of having an acute coronary syndrome(ACS)end up with a diagnosis of SC.According to reports,the morbidity of SC in the ICU patients ranges from 1.5to 28%,and the occurrence of SC and poor prognosis are associated with higher fatality rate.The pathogenesis of SC has not been fully clarified and may be related to catecholamine hypersecretion,low estrogen levels,coronary spasm,inflammation,and other factors.In recent years,there is growing evidence that inflammation plays an instrumental role in the development of SC.A disintegrin and metalloprotease 17(ADAM17),an important member of the ADAM family,plays a key role in the regulation of inflammation by splitting a variety of transmembrane proteins.It has been demonstrated that ADAM17 over expression also contributes to the development of SC.The cholinergic anti-inflammatory pathway(CAP),which consists of the vagus nerve and its transmitter acetylcholine,also plays an important role in the regulation of inflammation.Amiodarone(AD)is a cardiac multichannel blocker that shares the electrophysiological effects of class I,II,III,and IV antiarrhythmic drugs,but class III act primarily by blocking potassium channels.Clinically,AD has an important role in the treatment of tachyarrhythmias,especially atrial fibrillation patients with combined cardiac insufficiency.Some studies have demonstrated that AD has not only antiarrhythmic effects,but also anti-inflammatory effects.At present,it has not been investigated whether AD can exert cardioprotective effects by inhibiting the ADAM17 pathway and modulating the CAP to reduce the inflammatory response.Based on the above statement,in the present study,AD’s protective effects on cardiac function in SC rats were studied by using transthoracic echocardiography,enzyme-linked immunosorbent assay hematoxylin-eosin(HE)staining,and protein immunoblotting.To investigate the protective effects of AD on cardiac function in ISO rats from functional,morphological and protein expression aspects,and to investigate its mechanism of action,so as to provide new ideas for the treatment of SC.This study was divided into four parts.Part One Analysis of clinical characteristics and risk factors for death in patients with stress cardiomyopathy in the ICUObjective: We analyzed the clinical characteristics and risk factors for death of SC patients in the ICU of the Fourth Hospital of Hebei Medical University to find out the mortality risk factors,expecting to intervene the risk factors as early as possible to reduce the morbidity and mortality of SC in the ICU.Methods: This is a retrospective observational study that included 55 patients with SC admitted to the ICU of the Fourth Hospital of Hebei Medical University from April 2015 to March 2021.Two patients with previous atrial fibrillation were excluded,and 53 patients were finally included.According to whether the patients survived at 28 days or not,they were divided into survival group and non-survival group,and the clinical characteristics,laboratory tests and examinations of the two groups were compared.The variables with significant differences in the univariates were analyzed by logistic regression analysis.Therefore,we could screen for independent risk factors for death in SC patients in the ICU.Results: A total of 55 patients were included in this study,and 2 patients were excluded due to a medical history of atrial fibrillation,resulting in the inclusion of 53 patients.The percentage of males was 47.17%,of which88.68% of patients were induced by somatic factors,and the most common somatic stress trigger was sepsis(37.74%).Eighteen patients developed new-onset atrial fibrillation,representing 33.96%.The APACHE II score at the time of ICU admission was 21.17±8.41 and the SOFA score at the time of diagnosis of SC was 9.30±4.56.The 28-day morbidity rate of SC patients in the ICU was 64.15%.Univariate analysis showed that SOFA score at diagnosis of SC,eGFR <60 m L/min,maximum norepinephrine dose,new-onset AF and systolic heart failure were associated with poor prognosis(P<0.05)..Multifactorial analysis showed that independent risk factors for 28-day morbidity and mortality in patients with SC in the ICU were SOFA score at diagnosis of SC [OR,1.262;95%CI: 1.008-1.580;P=0.042],eGFR <60m L/min at diagnosis of SC(OR,6.049;95% CI: 1.224-29.897;P=0.027)and new onset atrial fibrillation(OR,7.789;95% CI: 1.063-57.088,P=0.043).Conclusions:1.Male SC patients in the ICU are common with a high percentage of somatic triggers.2.The 28-day morbidity and mortality rate of SC patients in the ICU is as high as 67.50%.3.New-onset AF is common in SC patients in ICU.4.New onset AF,SOFA score at diagnosis of SC,and eGFR <60m L/min at diagnosis of SC were independent risk factors for death in patients with SC.Part Two Stress-induced autonomic imbalance-induced inflammatory response is involved in the pathogenesis of stress cardiomyopathyObjective: To establish a rat model of SC and investigate the pathogenesis of SC.Methods: In this part of the experiment,male SD rats were selected and randomly divided into two groups,the control group(Control group,n=8)and the isoprenaline(ISO)induced SC group(ISO group,n=8).ISO group was given intraperitoneal injection of ISO 50mg/kg to replicate the SC model,and observing changes in left heart function by cardiac echography 2 hours later.Meanwhile,the levels of Cardiac troponin I(cTnI),brain natriuretic peptide(BNP),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),interleukin-18(IL-18),interleukin-1 β(IL-1 β),interleukin-18(IL-18),interleukin-1 β(IL-1 β),and the levels of norepinephrine(NE)and acetylcholinesterase(Ach)were measured by plasma.Then rats were executed and the heart tissues were reserved.The rats’ myocardial histomorphological changes were observed by HE staining.Results:1.After the intraperitoneal injection of ISO,the rats showed erect hair,depression,reduced activity,accelerated respiration and heart rate,and lying down in a congregated state.After 2h,which has statistically significant difference(P<0.01).Simultaneusly,some rats showed spherical changes in the apex of heart.2.Compared with the control group,plasma cardiac enzymes cTnI and BNP were elevated in the ISO group,with statistically significant differences(P<0.01).3.Compared with the Control group,the levels of plasma inflammatory factors IL-6,TNF-α,IL-18 and IL-1β were increased in the ISO group rats,with statistically significant differences(P<0.01).4.Compared with the Control group,the plasma NE levels of rats in the ISO group were significantly higher and the ACh levels were significantly lower,with statistical differences(P<0.05).5.Compared with the Control group,HE staining of myocardial tissues of rats in the ISO group showed that myocardial fibers were uniform,completely and and clearly structured,,and no inflammatory cell infiltration and cell necrosisConclusion:1.The administration of ISO intraperitoneally was able to successfully mimic the clinical SC model,with cardiac ultrasound,cTnI and BNP suggesting cardiac impairment.2.Plasma IL-6,TNF-α,IL-18,and IL-1β levels were increased after intraperitoneal injection of ISO.3.The administration of ISO intraperitoneally caused an increase in plasma NE levels and a decrease in Ach levels.Part Three Amiodarone can reduce cardiac dysfunction in stress cardiomyopathy ratsObjective: In this study,a rat model of SC was established by intraperitoneal injection of isoprenaline(ISO),and AD was administered after successful modeling to observe its effects on cardiac function in SC rats.Methods: Male SD rats were selected and given different doses(25,50,100 mg/kg)of AD after successful modeling,and finally 50 mg/kg was selected for subsequent experiments.The SD rats were divided into three groups,control group,ISO group and AD+ISO group.The left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)were observed by cardiac ultrasound in the three groups,and cTnI and BNP levels were measured by plasma sampling.The morphological changes of rat myocardium were observed by HE staining.Results:1.Effects of different doses of AD on the left heart function of ISO ratsCompared with the ISO group,the rats in the AD 25mg/kg+ISO group had no significant increase in LVEF and LVFS,which has statistically significant difference(P<0.05).However,there was no statistical difference in the LVEF and LVFS between the AD 50 mg/kg+ISO group and the AD100 mg/kg+ISO group.Among the four groups of rats: ISO group,AD 25mg/kg + ISO group,AD 50 mg/kg + ISO group and AD 100 mg/kg + ISO group,the highest survival rate was observed in the AD 50 mg/kg + ISO group and the highest mortality rate was observed in the AD 100 mg/kg + ISO group.2.Compared with rats in the ISO group,after administration of intraperitoneal AD 50 mg/kg for 4 h,the LVEF and LVFS in the AD + ISO group increased,with statistical differences(P<0.05).3.Compared to the ISO group,plasma cTnI and BNP concentrations were significantly lower in the AD + ISO group when AD 50 mg/kg was given intraperitoneally,with statistical differences(P<0.05).4.AD improved myocardial histomorphological changes in ratsIn comparison with Control group rats,myocardial fibers were observed to be incomplete and poorly aligned,and cell necrosis and infiltration of inflammatory and fibroblast-like cells were observed in ISO group rats after intraperitoneal injection of ISO 50 mg/kg for 6 hours.Intraperitoneal administration of AD 50 mg/kg showed a reduction in focal necrosis and inflammatory and fibroblast-like cell infiltration in myocardial cells and a significant decrease in myocardial tissue injury scores in the AD+ISO group,with statistical differences(P<0.05).Conclusion:1.AD can improve left heart function indexes,increase LVEF and LVFS,reduce cTnI and BNP levels,reduce myocardial injury and protect cardiac function in SC model rats.2.AD can reduce the pathological damage and inflammation in SC model rats.Part Four Amiodarone attenuates ISO-induced myocardial injury by inhibiting ADAM17 and enhancing cholinergic anti-inflammatory pathwaysObjective: In the third part of the study,we found that AD has a protective effect on the heart of SC rats.In this part of the study,we further evaluated whether AD exerts its myocardial protective effect by inhibiting the ADAM 17 pathway and enhancing the CAP to reduce the inflammatory response.Methods: Male SD rats were divided into three groups,Control group,ISO group and AD+ISO group.Intraperitoneal injection of AD or 5% glucose was given.The levels of TNF-α,IL-18,IL-1β and IL-6 in plasma and the levels of ACh and NE in plasma were measured by blood sampling after 6h.Meanwhile,the expression of ADAM17,p38 MAPKs,NF-ΚB,NLRP3,caspase-1 and α7n ACh R proteins in rat myocardial tissue were detected by western blotting.Results:1.Compared with the ISO group,the plasma NE level was significantly lower and the ACh level was significantly higher in the AD+ISO group,which was statistically different(P<0.05).2.Compared with the ISO group,the plasma IL-6,TNF-α,IL-18 and IL-1β levels were significantly decreased in the AD+ISO group rats,with statistical differences(P<0.05).3.Compared with the ISO group,the protein expression of ADAM17,p38 MAPKs,NF-ΚB,NLRP3 and caspase-1 was decreased and that of α7n ACh R was increased in the heart tissues of AD+ISO rats,with statistical differences(P<0.05).Conclusions:1.AD can reduce plasma NE levels and increase plasma Ach levels.2.AD can reduce the inflammatory response in SC rats.3.AD attenuates inflammatory response by inhibiting ADAM17,NLRP3,caspase 1 protein expression and enhancing CAP in SC rat heart tissue.