| BackgroundWith the rapid development of science,technology,and medical care,people’s awareness of health and disease prevention is increasing.With the change of living habits and lifestyles,the problems of hypertension,diabetes,hyperlipidemia and population aging continue to emerge,and the incidence of cardiovascular disease is increasing year by year.Myocardial infarction(MI)is pathologically defined as myocardial cell necrosis caused by persistent blood flow and nutrient disruption,which is one of the highest mortality rates in the clinic.And malignant arrhythmia is the chief culprit of sudden cardiac death(SCD).It is currently believed that the occurrence of ventricular arrhythmias(VAs)after MI were mainly related to sympathetic activation,inflammatory aggregation,cardiac electromechanical remodeling,ion-channels changing and catecholamines releasing.Although advanced medical technologies such as emergency PCI,thrombolysis,ICD implantation and radiofrequency ablation have been widely used in clinical hospitals;the establishment of chest pain centers also provides a good platform for AMI patients to open criminal blood vessels as soon as possible.However,an average of 4.8-46 person/year/1 million people died of SCD.In clinic,antiarrhythmic drugs have their application restrictions,contraindications and arrhythmogenic effects,such as cordarone,has toxic effects of liver and thyroid function,and also lead to the prolongation of QT interval.So VAs Prevention and treatment is still the focus of current clinical work.Animal experimental studies have found that ganglion block,renal sympathetic denervation and other techniques can effectively reduce peripheral sympathetic tone,while reducing the incidence of VAs post-MI,which could effectively alleviate the symptoms of about 60%patients with intractable angina pectoris.And this surgical treatment has also been applied to clinical practice and achieved some clinical results,but it has not been fully carried out at present.Therefore,sympathetic tone regulation may become an important target for the prevention and treatment of malignant VAs after MI.Central nervous system(CNS),)the most advanced nerves center in human body,is involved in the regulation of pathophysiological processes of various diseases.It was well recognized that the CNS regulates the peripheral sympathetic nervous system.Hypothalamic paraventricular nucleus(PVN)is located on both sides of the superior of the third ventricle in hypothalamic,and is one of the most significant nuclei in the anterior hypothalamic area and the regulatory center of autonomic nerve function and cardiovascular activity.Studies have shown that the activity of sympathetic neurons in the PVN is significantly activated in heart failure rats and spontaneously hypertensive rats,which in turn activated the peripheral sympathetic nervous system such as elevated blood pressure,plasma norepinephrine(NE)level and renal sympathetic activity(RSNA).It has been reported that PI3K/AKT pathway was activated and involved in the regulation of sympathetic neuron activity.PI3K/AKT signaling pathway is one of the important intracellular signal transduction pathways.In spontaneously hypertensive rats,PI3K/AKT signaling pathway in the PVN was activated and involved in the regulating of sympathetic neurons activity.However,inhibition this signaling pathway can reduce peripheral sympathetic activity and effectively reduce blood pressure,but the specific mechanism remains unclear.Therefore,we hypothesize that the PI3K/AKT signaling pathway in the PVN plays an important role in the regulation of sympathetic nerve activity after MI,and targeted intervention of the pathway can effectively reduce the incidence of malignant arrhythmias after MI.ObjectiveThe aim of this study was to clarify the expression level of PI3K/AKT signaling pathway in the PVN after acute MI,and also to evaluate the infarct size,cardiac function,NE level,RSNA and arrhythmia susceptibility and other related indicators in rats by inhibiting PI3K activity with LY294002(PI3K inhibitor),in order to investigate the regulation mechanism of PVN on peripheral sympathetic activity after MI.Methods1,Animal modelAfter anesthesia with 3%pentobarbital sodium,a stainless-steel gauge guide cannula was stereotaxically implanted into the PVN by brain stereotaxic apparatus,and fixed by dental cement.Antibiotics were injected during the period to prevent infection.After 7 days of recovery,the rats were randomly divided into 4 groups:group A:sham operation + Dimethyl sulfoxide(DMSO),sham operation,proximal anterior descending coronary artery without threading;group B:sham operation +LY294002;Group C:MI + DMSO,MI model,ligation of the proximal anterior descending coronary artery;Group D:MI + LY294002.After MI surgery,DMSO or LY294002(5 nmol/L)was microinjected into the PVN after the MI surgery according to the experimental design with a 100-nl volume for each PVN and was removed slowly after 5 min every 12 h for 7 days.2,Analyze the arrhythmia susceptibilitySeven days after MI,before the rats were sacrificed,the induction rate of arrhythmias and the score of arrhythmia susceptibility were measured by programmed electrical stimulation program with ventilator support.3,hemodynamics experimentAfter the microcatheter was inserted into the carotid artery of the rat,heart rate(HR),LVSP(left ventricular systolic pressure),LVDP(left ventricular diastolic pressure),EF(ejection fraction),+dp/dtmax(maximal slope of the systolic pressure increment),-dp/dtmin(maximal slope of the diastolic pressure decrement)was obtained by Power Lab system.4,RSNA analysisIn the right lying position,the left waist and abdomen were cut longitudinally,and the kidney was exposed,then the renal sympathetic nerve was separated to measure the RSNA.5,blood pressure measurementThe systolic and diastolic blood pressure of the caudal artery was measured by the tail sleeve blood pressure meter three times,and the average value was measured.6,ElisaThe supernatant was collected before the rats were killed,and the supernatants were collected from the periinfarction tissue homogenate of the rats,the changes of NE level were detected by Elisa.7,Immunohistochemistry experimentAfter the rats were perfused with paraformaldehyde,the brain tissue and heart tissue were extracted immediately and paraffin sections were prepared.Masson test showed that the MI model was successful.And the infarction area of rats was measured,the infarction area of 30%-50%were enrolled.The expression of Fra-LI positive neurons in the PVN were detected by immunohistochemistry after brain tissue sections8,Western BlotThe PVN tissues of each group were taken,and the protein expression of p-PI3Kp85,total-PI3K,p-AKT,total-AKT and GAPDH in the PVN was detected.Results1,LY294002 microinjection reduced the sympathetic activity in the PVN.Immunohistochemical staining showed that the densities of Fra-LI immunoreactivity in the PVN in MI groups were higher compared with sham groups,and could be attenuated significantly in LY294002-treated rats compared with MI+DMSO group(128±4.68 vs 197.4±14.4,p<0.05).The possibility of the PVN damage caused by the microinjection has been already excluded.2,Effect of LY294002 microinjection on hemodynamicsCompared with the two sham groups,the +dp/dtmax,-dp/dtmin and EF in MI+DMSO group and MI+LY294002 group was significantly decreased,but there was no significant difference between the two MI groups.And the heart rate(HR)was increased significantly in MI groups compared with the sham groups,the HR in MI+LY294002 group was significantly higher than that in MI+DMSO group.3,LY294002 microinjection improved peripheral sympathetic activityThe plasma NE levels and myocardial NE levels were significantly upregulated in the MI+DMSO group compared to the sham groups(plasma NE,89.42±11.21 pg/ml vs 26.61 ± 1.03 pg/ml,25.53±0.93 pg/ml;myocardial NE,27.36±2.08 pg/mg vs 14.09±1.20 pg/mg and 13.74±1.74 pg/mg,p<0.05).The NE levels were significantly lower in the MI+LY294002 group than in the MI+DMSO group(plasma NE,37.17±3.73 pg/ml vs 89.42±11.21 pg/ml;myocardial NE,20.01±1.45 pg/mg vs 27.36±2.08 pg/mg,p<0.05).There was no significant difference in RSNA baseline between the two sham groups,but the RSNA baseline of the MI+DMSO group was significantly increased compared with that in the sham groups(0.13±0.01 uv.s vs 0.06±0.01 uv.s and 0.06±0.01 uv.s,p<0.05),indicating that MI operation resulted in peripheral sympathetic nerves overactivition.And the LY294002 treatment MI group had a significantly reduced RSNA baseline compared with the MI+DMSO group(0.09±0.01 uv.s vs 0.13±0.01 uv.s,p<0.05).4,LY294002 microinjection attenuated arrhythmia susceptibilityThe inducibility of ventricular tachyarrhythmia in sham group + DMSO,sham group + LY294002,MI+DMSO and MI+LY294002 were 12.5%,12.5%,75%and 40%,respectively.The score of arrhythmia susceptibility in the two sham groups was close to 0,and that in MI+DMSO group was about 3.3.However the rats in MI+LY294002 group were about 1.4,and the results showed that arrhythmia susceptibility was significantly increased after MI,and the induction rate of arrhythmia was decreased after LY294002 microinjection.5,PI3K/AKT pathway protein expression in the PVNThe protein relative level of PI3K and AKT were significantly higher in the PVN of MI rats,as indicated by a 2-fold increase in the p-PI3K-to-tota-PI3K ratio(MI+DMSO vs sham+DMSO and sham+LY294002:0.79±0.06 vs 0.40±0.04 and 0.44±0.03,both p<0.05,)and a 1.5-fold increase in the p-AKT-to-total-AKT ratio(0.88±0.05 vs 0.59±0.06 and 0.53±0.03,both p<0.05),manifesting that MI significantly activated the PI3K-AKT pathways in the PVN.LY294002 treatment group in MI rats resulted in a significant reduction in the relative p-PI3K-to-total,PI3K ratio of 0.58±0.04 compared with 0.79±0.06 for the relative p-PI3K-to-total-PI3K ratio in the MI+DMSO group.Treatment with LY294002 decreased the p-AKT-to-total-AKT ratio(0.61±0.01vs 0.88±0.05,p<0.05)in theMI+LY294002 group compared with the MI+DMSO group.ConclusionThe sympathetic neuron activity in the PVN and peripheral sympathetic nervous system were overactivated post MI,and targeted inhibition of the sympathetic output in the PVN via PI3K-AKT pathway may be a main mechanism in regulation of peripheral sympathetic activity and VAs.BackgroundThe superior cervical ganglion(SCG)is the largest sympathetic ganglion in the cervical sympathetic trunk.After the metastasis,the neurons in the spinal cord segment emit postganglionic nerve fibers to innervate the corresponding tissues and organs.The superior cardiac sympathetic nerves,the middle cardiac nerve and the inferior cardiac sympathetic nerves from the the SCG,middle cervical ganglion and stellate ganglion form the cardiac plexus,which is involved in the regulation of the cardiac autonomic nervous system function.SCG was involved in the feedback regulation of cardiac autonomic nerve after MI.At present,ganglion ablation and block have been used in the treatment of refractory angina pectoris and can alleviate 60%of angina pectoris symptoms,but the specific mechanism remains unclear.γ-aminobutyric acid(GABA)is an important inhibitory neurotransmitter in the central nervous system,which is involved in the regulation of various psychiatric diseases and cardiovascular diseases,such as Parkinson’s disease,anxiety,hypertension,arrhythmia and so on.GABA plays an inhibitory role mainly by binding to ionic GABAA receptor(GABAAR)and GABAB receptor.GABA and excitatory neurotransmitters in the PVN coordinate and antagonize each other to regulate cardiac autonomic nerve activity.GABA dysregulation in the PVN is closely related to the occurrence of cardiovascular diseases such as hypertension,heart failure and arrhythmia.At the same time,the up-regulation of GABAAR expression in special nuclei of the brain could decrease sympathetic activity.In recent years,studies have found that the GABA system plays an important physiological role in peripheral tissues and organs(such as liver,pancreas,gastrointestinal tract,fallopian tube,and cardiovascular system).Exogenous GABA(oral administration)could reduce the blood pressure by an average of 20.8±3.5mmHg in spontaneously hypertensive rats,but had no significant effect on normal rats,which may be associated with abnormally increased of sympathetic tone in spontaneously hypertensive rats,which was suggested that peripheral GABA has a clear regulation of blood pressure.Intravenous injection of GABA could block the premature ventricular contractions and VT induced by aconitine,indicating the cardiovascular regulation of the GABAergic system in the peripheral nervous system.Previous studies have confirmed the expression of GABAergic signaling system in thr SCG,but currently there is a lack of physiological and pathological studies on GABAergic systems.In this study,we investigated the role of GABAergic signal system on sympathetic nerve activity and the underlying mechanism.Objective1)To investigate the regulation of exogenous GABA on cardiovascular function and whether GABAergic signaling system in the SCG was involved in the regulation of sympathetic nerve activity after myocardial infarction.2)To determine whether GABAergic system regulates NE release through P2X7/AKT pathway to provide intervention targets for the development and treatment of new clinical drugs.MethodsProtocols 1For the antiarrhythmic effects of exogenous GABA experiment,rats were completely randomized into the following three groups:group A,sham surgery;group B,MI;group C,MI + exogenous GABA.GABA(Sigma)was dissolved in water for rats to drink(500mg/kg)from one day before the experimentation until the end of the experiment.Rats in group A and B received water only without adding any drugs.The anterior descending coronary artery was ligated for MI model and the sham operation group was not ligated.Seven days after MI,the arrhythmia susceptibility was determined by programmed electrical stimulation in three groups.The hemodynamic index was measured by small animal ventricular pressure volume system,the heart rate variability was detected by small animal telemetry,and the sympathetic activity was observed by renal sympathetic never activity(RSNA).Paraffin sections were stained with masson to measure infarct size(30-50%of the infarct size was selected to meet the inclusion criteria).The expression of GABAAR was measured by Western Blot in the SCG.The changes of NE were measured by Elisa in peripheral blood and infarcted myocardium.Protocols2For GABAARβ2 knockdown experiment to explore the GABAergic signal system in regulation of arrhythmia after MI,the GABAARβ2 shRNA virus was first prepared and injected into the bilateral SCG by microinjector.After 4 days,SCG was isolated to observe whether the virus was effectively infected.The efficacy of the virus was detected by qRT-PCR,and the experiment was preferably selected according to the above experimental results.And then rats were randomly divided into four groups,sham surgery + vehicle;sham surgery+ GABAARβ2 shRNA lentiviral vector;MI +vehicle;MI + GABAARβ2 shRNA lentiviral vector.Vehicle and GABAARβ2 shRNA lentiviral vector were targeted microinjection into SCG four days before MI surgery.Four days aftr microinjection,MI modle was processed.Before death on day 7 post-MI,electrophysiological testing and RSNA were performed,and Heart tissues,blood samples and SCG were then collected for Western blotting,qRT-PCR,immunohistochemistry,and ELISA.Protocols3For the underline molecular mechanism of GABAergic system on the antiarrhythmia effect,sympathetic neurons isolated from fetal SCG were used.The distribution and localization of P2X7 receptor(P2X7R)and GABAARβ2 in TH-positive neurons were processed by immunofluorescence staining.In this protocol,Ca2+ imaging was performed by different treatments such as ATP,GABA receptor agonist(muscimol)+ ATP,GABA receptor antagonist(SR 95531)+ muscimol + ATP.Before and after different administration,488nm excitation light was selected to detect changes of intracellular Ca2+Results1,Masson’s trichrome,electrocardiogram,gross view of the heart were used as a criterion for successful MI model.Only 30-50%infarcted rats were enrolled,and there was no significant difference in infarction area between MI group and MI+GABA group.The present study determined that the plasma NE levels and myocardial NE levels in the MI group were significantly higher than those in the sham group,and the levels of NE were significantly significantly reduced after GABA intervention.Programmed electrical stimulation was performed.The inducible VAs in the sham group was only 10%,and the susceptibility score was close to 0.The inducible VAs in the MI group was 80%,and the susceptibility score was 3.1±0.8,compared with 50%inducible VAs in MI+GABA group.And also the susceptibility score was significantly lower than that in Ml group(1.4±0.5 VS 3.1±0.8,P<0.05).RSNA test also found that RSNA scores and baseline levels were abnormally elevated in MI rats,and those in MI+GABA group were significantly lower compared with MI group.Peripheral NE,RSNA reflected the overactivation of peripheral sympathetic activity after MI,and exogenous GABA can effectively improve the sympathetic overactivation.Hemodynamic dates were shown that the dP/dt and EF values in the MI+GABA group are significantly higher than those in MI group,which indicated that GABA could improve cardiac function in rats.The expression level of GABAARβ2 in SCG was increased of MI+GABA group compared with MI group,suggesting that exogenous GABA may play a role via GABAARβ2 in SCG.2,To define the role of GABAergic system in regulation of sympathetic activity,After preparation of GABAARβ2 shRNA,the most potent virus was selected according to the results of RSNA,Western Blot and qRT-PCR.After MI,the related experiments showed that the mortality of the MI+ GABAARβ2 shRNA group was significantly higher than that in the MI+Vehicle group.The electrocardiogram showed that the rats died of malignant arrhythmia.After MI 7 days,the relevant experiments were carried out.The results showed that the basic level of NE,RSNA and arrhythmia susceptibility score in MI+ GABAARβ2 shRNA group were significantly higher than those in MI+Vehicle group.The result indicates that the GABAergic system in SCG was involved in the regulation of cardiovascular function post-MI.3,SCG immunofluorescence double staining suggested that the expression of GABA synthase GAD65/68 was mainly located on the cell body of TH-positive sympathetic neurons,suggesting that TH-positive neurons can also synthesize GABA.Cellular immunofluorescence was performed and the results showed that P2X7 receptor and GABAARβ2 were mainly located on the cell bodies and axons of TH-positive neurons.Calcium imaging results showed that after ATP treatment,the concentration of Ca2+ in the neurons was significantly increased,which could be inhibited by the muscimol(GABAR agonist).After adding SR 95531(GABAR inhibitor)+ muscimol + ATP,which can antagonize the inhibition of muscimol,and the intracellular Ca 2+ concentration was significantly increased,indicated that GABA participates in regulation of the intracellular Ca2+ concentration in sympathetic neurons through P2X7/AKT signaling pathway.ConclusionGABAergic signaling system is expressed in the SCG.Systemic administration of GABA could reduce peripheral sympathetic activity and the incidence of arrhythmia after MI.The GABAARβ2 knockout in the SCG worsened the high level of RSNA,NE,and arrhythmia susceptibility scores caused by MI,further confirming that the GABAergic system in the SCG was involved in the regulation of sympathetic activity.We confirmed that GABA regulated peripheral sympathetic activity by inhibition of P2X7R,thereby reducing intracellular Ca2+ influx and NE release.Therefore,the GABA system regulates NE release through the P2X7/AKT pathway,thereby reducing the incidence of arrhythmia after MI and providing a potential target for the prevention and treatment of clinical arrhythmias. |
PDF Full Text Request