Protective Effects Of PGE1 And NAD~+ On Post-cardiac Arrest Myocardial Dysfunction

Cardiac arrest(CA)is one of the most critical symptoms in clinical,as is hitting the person at all ages without any pre-signal.Statistics reveal that 8-9 million victims undergo CA worldwide each year.Considering its largest population,China has the most sufferers of CA in the world.Although,over the last half-century,cardiopulmonary resuscitation(CPR)has been making progress,which increases the rate of return of spontaneous circulation(ROSC)up to 20%,yet the hospital discharge remains below 2%.Postcardiac arrest syndrome(PCAS)refers to the constellation of abnormalities that develops after ROSC from CA,including post-cardiac arrest myocardial dysfunction(PAMD),neurological dysfunction,etcetera.Deaths within the first 24 hours after ROSC are typically caused by PAMD,while later deaths result from neurological dysfunction.Therefore,mitigating PAMD is a crucial part of improved survival to hospital discharge with favorable outcomes.Despite lacking a clear understanding,recent literature finds that microcirculation dysfunction and mitochondrial dysfunction are involved in the mechanisms of PAMD.After ROSC,mean arterial pressure(MAP)would be able to sustain the perfusion of coronary,but microcirculation dysfunction severely weakens the exchange of interstitial fluid in heart.In that case,coronary perfusion pressure(CPP)is not closely related to the transfer of interstitial fluid anymore.In addition,mitochondrial dysfunction further impairs cellular energy metabolism.These factors eventually lead to cardiomyocyte apoptosis.Hence,it is rational to consider microcirculation dysfunction and mitochondrial dysfunction as therapeutic targets.Prostaglandin E1(PGE1)is one member of prostaglandin family naturally existing in the human body,which is extensively used for treating microcirculation dysfunction and peripheral ischemia.Besides,recent studies also indicate that PGE1 could alleviate mitochondrial dysfunction in ischemia-reperfusion injury.Based on these factors,we speculated that PGE1 would be a potential treatment for PAMD since intervening in the microcirculation and mitochondrial dysfunction.However,the relationship between PGE1 and PAMD is still unknown.Therefore,in the present study,we aimed to investigate the protective effects and mechanisms of PGE1 on PAMD in a rat model of CA and a H9c2 cell model of ischemia-reperfusion injury.ObjectivesThe main aims of the present study are:1.To investigate the protective effects of PGE1 against PAMD.2.To investigate whether alleviating microcirculation dysfunction is involved in the mechanisms of PGE1 against PAMD.3.To investigate whether attenuating mitochondrial dysfunction is involved in the mechanisms of PGE1 against PAMD.Methods(?)In vivo Study1.Asphyxia-induced CA model establishmentMale Wistar rats,weighing 380-430 g,were anesthetized with isoflurane.Rats underwent 8 min of asphyxia followed by CPR.Only rats that achieved ROSC within 10 min were used for further study.2.Experimental groups and drug administrationRats were randomly allocated to three groups after anesthetized,each group would be divided into 2 subgroups:4 h(n=7 per group)and 72 h(n=10 per group):?CA+PGE1 group:rats were intravenously treated with PGE1(1 ?g/kg)at the onset of ROSC;?CA group:rats were intravenously treated with vehicle(saline)at the onset of ROSC;?Sham group;rats underwent the same surgical procedure without asphyxia or CPR.Rats in 4 h subgroup were sacrificed and the tissues were collected at 4 h after ROSC.Rats in 72 h subgroup were observed up to 72 h after ROSC.3.Heart rate and MAP monitoringElectrocardiogram was used to measure heart rate(HR).A catheter is advanced into the left femoral artery for measurement of MAP.4.Assessment of myocardial functionLeft ventricular ejection fraction(LVEF)and cardiac output(CO)at baseline,1 h,2 h,3 h,and 4 h after ROSC were measured by echocardiography.5.Sublingual microcirculationSublingual microcirculation was visualized at baseline,1 h,2 h,3 h and 4 h after ROSC.Microvascular flow index(MFI)and Perfused microvessel density(PVD)were quantitated.6.Terminal deoxynucleotide nick-end labeling(TUNEL)assayCardiomyocyte apoptosis in rats was detected by a TUNEL assay at 4 h after ROSC.7.The levels of TNNI3 in the plasmaTNNI3 in the plasma at 4 h after ROSC was assayed by Elisa kit.8.Electron microscope observationMitochondria in rat heart were examined in a transmission electron microscope at 4 h after ROSC.9.Measurement of TNF-? and IL-6Tumor necrosis factor ?(TNF-?)and IL-6(interleukin-6,IL-6)in plasma and heart tissue at baseline and 4 h after ROSC were measured by Elisa and Western-blot respectively.10.Mitochondrial permeability transition pore(mPTP)openingFresh mitochondria were isolated form hear tissue at 4 h after ROSC.mPTP opening was measured using a spectrophotometry kit.11.Mitochondrial ROS assayFresh mitochondria were isolated form hear tissue at 4 h after ROSC.ROS in mitochondrial was detected by a fluorescence quantitative kit.12.Protein expression of GSK3?,cytochrome c and caspase-3Protein levels of GSK3? and caspase-3 in heart tissue at 4 h after ROSC were detected by western-blot.Cytoplasmic protein without mitochondria was used to measure cytochrome c expression.13.Survival analysisAll catheters were removed and wounds were surgically closed at 4 h after ROSC.The rats were closely monitored during 72 h.(?)In vitro Study1.H9c2 hypoxia-reoxygenation(H/R)cell model establishmentH9c2 cells in no-glucose DMEM were exposed to 1%O2,94%N2,and 5%CO2 for 12 h to mimic ischemia.Then,the medium was replaced with high-glucose DMEM and the cells were transferred to a regular incubator(95%air,5%CO2,37?)for 12 h to mimic reperfusion.2.Experimental groups and drug administrationCells were divided into three groups:(1)H/R+PGE1 group:cells were treated with PGE1(0.5uM)at the end of hypoxia and no-glucose phase.(2)H/R group:cells underwent H/R process without PGE1 treatment.(3)Control group:cells were cultured in a regular incubator.3.TUNEL assayApoptosis in three groups were detected by TUNEL.4.Measurement of mPTP openingFresh mitochondria were isolated from counting cells.mPTP opening was measured by a spectrophotometry kit and an immunofluorescence method.5.Mitochondrial ROS assayFresh mitochondria were isolated form H9c2 cells.ROS in mitochondrial was detected by a fluorescence quantitative kit.6.Protein expression of GSK3?,cytochrome c and caspase-3Protein levels of GSK3?,cytochrome c and caspase-3 in H9c2 cells were detected by Western-blot.Results1.PGE1 ameliorates cardiac dysfunctionThe results showed that CO and EF were significantly impaired within 4 h after ROSC in the CA group when compared to the sham group but were significantly preserved by PGE1 treatment.2.PGE1 improved 72 h survival rateThe survival rates were monitored for 72 h.Kaplan-Meier survival curves indicated a rapid decline in the survival rate of rats in the CA group within 24 h following ROSC.The survival rate in the CA+PGE1 group was significantly higher than that in the CA group3.PGE1 attenuated microcirculation dysfunctionBuccal microcirculation was significantly poorer within 4 h after ROSC in the CA group than that in sham group.PGE1 treatment attenuated the microcirculation dysfunction.4.PGE1 decreased TNF-? and IL-6The level of TNF-? and IL-6 were higher in plasma within 4 h after ROSC in CA group compared to sham group.PGE1 treatment significantly decreased TNF-? and IL-6.Results in heart tissue were consistent with those in plasma.5.PGE1 suppresses cardiomyocyte apoptosisIn vivo,the level of apoptosis in the CA group was significantly increased when compared to that in the sham group.In contrast,cardiac apoptosis decreased after treatment with PGE1.In vitro,the fraction of apoptotic H9c2 cells in the H/R+PGE1 group was significantly smaller than that in the H/R group.In addition,PGE1 treatment significantly decreased the level of TNNI3 in the plasma than that in the CA group.6.PGE1 relieved damages in mitochondrial ultrastructureMitochondrial was significantly swelled in cardiomyocytes in CA group compared to sham group.PGE1 treatment relieved the mitochondrial swelling induced by CA.7.PGE1 blocks mPTP openingIn vivo,the mPTP opening was increased in the CA group compared to the sham group.PGE1 treatment reduced mPTP opening.In vitro,mPTP opening was blocked by PGE1 treatment.8.PGE1 regulates GSK3?,cleaved caspase-3 and cytochrome c expressionIn vivo,GSK3? phosphorylation decreased in the CA group compared with the sham group GSK3? phosphorylation was higher in the CA+PGE1 group than in the CA group.In vitro,GSK3? phosphorylation was decreased in the H/R group,compared with the control group.However,it was partly restored following PGE1 treatment.The cytoplasmic protein levels of cleaved caspase-3 and cytochrome c were significantly increased in the CA group in vivo,compared with the sham group.PGE1 treatment partially suppressed these effects.The results obtained in vitro were consistent with the in vivo data.Indeed,PGE1 treatment inhibited the increase in the expression levels of cleaved caspase-3 and cytochrome c induced by H/RConclusion1.PGE1 treatment at the onset of ROSC attenuated PAMD and improved the survival rate after CA.2.Its benefits were partially attributed to alleviating microcirculation.2.Inhibiting mPTP-cytochrome c-caspase3-apoptosis by GSK3? pathway,not ROS,was involved in the protective mechanisms of PGE1.Cardiac arrest(CA)is still a huge public health burden.Post-resuscitation cardiac dysfunction(PAMD)is one of the main causes of early death in CA patients after resuming spontaneous circulation(ROSC).Due to its complicated pathogenesis,there is no effective treatment method in clinical practice.Therefore,finding new treatment methods has great practical significance for improving the survival rate of CA patients.In previous studies,it has been reported that the dysfunction of the mitochondrial respiratory chain in cardiomyocytes after ROSC,which in turn decreases the production of adenosine triphosphate(ATP),is one of the mechanisms of PAMD.Among them,the impairment of the oxidative phosphorylation ability of the respiratory chain complex I(Complex ?)of cardiomyocytes after ROSC is a key target.The decline of Complex I's oxidative phosphorylation capacity may be the key cause of the decreased ATP synthesis after ROSC.However,the mechanism of the decline of Complex I oxidative phosphorylation is still unclear,and there is no effective intervention to alleviate it.Nicotinamide adenine dinucleotide(NAD+)is an important coenzyme in mitochondria.After CA,the level of NAD+ in mitochondria is significantly reduced.NAD+has multiple roles in the body.In recent years,it has attracted much attention because of its ability to regulate the activity of silent information regulators(SIRTs).SIRT3 is a type of diacetyl protease anchored in the mitochondria.Its activity depends on NAD+.It is also the main regulator of protein acetylation in the mitochondria.Recent studies have shown that SIRT3 can affect oxidative phosphorylation by regulating the acetylation of complex I subunit proteins,thereby affecting mitochondrial ATP synthesis.NDUFA9 is the main subunit responsible for oxidative phosphorylation in Complex I.It has been reported that acetylated NDUFA9 can down-regulate the oxidative phosphorylation capacity of Complex I.This suggests that NAD+-SIRT3-NDUFA9 acetylation may mediate the reduction of the oxidative phosphorylation function of Complex I after CA and participate in the formation of PAMD.In addition,there are studies showing that intravenous supplementation of exogenous NAD+can improve neurological function in a model of focal cerebral ischemia.Therefore,this section of the study hypothesized that intravenous administration of NAD+may alleviate PAMD,and its mechanism may involve SIRT3-NDUFA9 acetylation.ObjectivesThe main aims of the present study are:(1)to investigate the protective effects of NAD+on post-cardiac arrest neurological dysfunction.(2)to investigate whether NAD+-SIRT3-NDUFA9 acetylation is involved in the mechanisms of NAD+against post-cardiac arrest neurological dysfunction.Methods1.Ventricular fibrillation-induced CA model establishmentMale SD rats,weighing 450-550 g,were anesthetized with pentobarbital.Rats underwent 8 min of asphyxia followed by CPR.The duration of the cardiac arrest was 6 minutes.Electrical defibrillation was given after 8 minutes of mechanical ventilation and chest compressions.Rats that reached ROSC within three times of electrical defibrillation were included in the group to continue the experiment.2.Experimental groups and drug administrationRats were randomly allocated to three groups after anesthetized:?NAD group:rats were intravenously treated with NAD+(20 mg/kg)at the onset of ROSC;?Control group:rats were intravenously treated with vehicle(saline)at the onset of ROSC;?Sham group:rats underwent the same surgical procedure without ventricular fibrillation or CPR.3.Heart rate and MAP monitoringElectrocardiogram was used to measure heart rate(HR).A catheter is advanced into the left femoral artery for measurement of MAP.4.Assessment of cardio functionCardio function within 4 h after ROSC were measured by Ultrasound.5.Detection of ATP levels in rat myocardial tissueThe rats were sacrificed 4 h after ROSC.After homogenization,the ATP level was measured with a microplate reader.6.Detection of mitochondrial respiration activity in rat myocardial tissueThe myocardial tissue of the left ventricle was harvested at 4 h after ROSC.After homogenization,the respiration activity of Complex ?-? were measured by high-resolution respirometry.7.Detection of NAD+levels in mitochondria of rat myocardial tissueThe myocardial tissue of the left ventricle was harvested at 4 h after ROSC and mitochondria were purified.The NAD+levels in the mitochondria were detected by a microplate reader.8.Protein expression of SIRT3,acetylated NDUFA9 in rat myocardial tissueThe myocardial tissue of the left ventricle was harvested at 4 h after ROSC and mitochondria were purified.Protein levels of SIRT3 and acetylated NDUFA8 was detected by Co-IP and Western-Blot.9.Survival analysisall catheters were removed and wounds were surgically closed at 4 h after ROSC.The rats were closely monitored during 72 h.Results1.NAD+ improved MAP and cardio functionThe MAP.of the NAD group was significantly higher than that of the Control group within 1-4 h after ROSC.The ultrasound results showed that the ejection fraction(EF),cardiac output(CO),and cardiac index(MPI)of the NAD group were significantly better than those of the Control group.2.NAD+ administration increased the ATP levelsCompared with the Sham group,the ATP level in the myocardial tissue of the control group rats was significantly lower at 4 h after ROSC.The ATP level in myocardial tissue of rats in the NAD group was significantly higher than that in the control group at 4 h after ROSC.3.NAD+ improved mitochondrial respiration activityCompared with the Sham group,the respiration activity of Routine and Complex I of the control group rats were impaired at 4 h after ROSC.NAD+ treatment improved that.4.NAD+ increased the level of NAD+in the mitochondriaCompared with the Sham group,the NAD+ level in the myocardial mitochondria in the Control group was significantly reduced at 4 h after ROSC.The NAD+ level in the NAD group was higher than that in the Control group.5.NAD+ increased the expression of SIRT3 and down-regulated the expression of acetylated NDUFA9Compared with the Sham group,the level of mitochondrial protein acetylation in the Control group increased,the expression of acetylated NDUFA9 increased,and the expression of SIRT3 decreased.The level of mitochondrial protein acetylation in NAD group was significantly decreased,the expression of acetylated NDUFA9 was lower,and the expression of SIRT3 was higher than that of Control group.6.NAD+ improved 72 h survival rateThe survival rates were monitored for 72 h.The survival rate in the NAD group was significantly higher than that in the Control group.Conclusion1.NAD+ treatment at the onset of ROSC attenuated PAMD.2.The oxidative phosphorylation capacity of Complex I regulated by SIRT3-NDUFA9 acetylation was involved in the protective mechanisms of NAD+.