Effects And Mechanisms Of P2X₇ Receptor On Sympathetic Remodeling And Cardiac Function In Rats With Myocardial Infarction

Myocardial infarction?MI?is a series of pathophysiological changes,following unstable coronary atherosclerotic plaque rupturing,the formation of thrombosis and complete occlusion of coronary artery.It is a serious kind of coronary atherosclerotic heart disease?coronary heart disease,CHD?.The morbidity and lethality of MI is gradually increasing,which brought great economic burden to the development of global public health.Modern research shows that an important factor which threaten the survival and prognosis of patients after myocardial infarction is the complications such as arrhythmia,heart failure and so on.About 50%patients died of the fatal ventricular arrhythmia?VAs?after myocardial infarction.The occurrence of ventricular arrhythmia post myocardial infarction is the result of a variety of pathophysiological factors such as electrical remodeling,neuronal remodeling,multiple cytokines and gene expression.With the intensive study of myocardial infarction,the relationship between sympathetic remodeling and ventricular arrhythmia has aroused the attention of the majority of scholars.In the early stage,there was a low degree of dominance of the sympathetic nerve.In the later stage,Sympathetic nerve regenerate,there was a significant difference in the density and function of the sympathetic nerve in the area of myocardial infarction.This acts as the nerve matrix of ventricular arrhythmia,and thus easily lead to fatal ventricular arrhythmias.In addition,over-activation of Sympathetic nerve led to increased secretion of catecholamines active,increased cytotoxicity,myocardial cell apoptosis,necrosis,also had a certain impact on cardiac function.Therefore,MI sympathetic remodeling plays an important role in the development of ventricular arrhythmias and changes of cardiac function,and research of its mechanism has positive clinical significance.At present,massive evidences reveal that inflammation and associated cytokines play a key role in stimulating axonal growth and regeneration?sympathetic remodeling?.P2X₇ receptor is widely distributed in many organs,tissues and cells,P2X₇ receptor is expressed in a variety of inflammatory pathology,and involved in the regulation of the release of inflammatory factors.It has been confirmed that P2X₇ receptor is closely related to a variety of inflammatory diseases.Antagonizing the P2X₇ receptor has a certain therapeutic effect on inflammatory diseases,In addition,a large number of studies have shown that P2X₇R is involved in the regulation of cerebral cortical neuronal injury process,which can improve neurodegeneration,In addition P2X₇R plays an important pathophysiological function in central nervous system disorders?including ischemic stroke,subarachnoid hemorrhage,epilepsy,neuropathic pain and neurodegenerative diseases?.However,whether P2X₇R participates in sympathetic remodeling after myocardial infarction has not yet been studied.It has been confirmed that activation of P2X₇R stimulates the phosphorylation of cellular signaling molecules,including extracellular signal-regulated kinase?ERK1/2?and p38 mitogen-activated protein kinase?MAPK?,triggering reactive oxygen production,nuclear factor kappaB?NF-?B?activation.NF-?B is a central regulator of multiple immune responses and is a ubiquitous transcription factor.Under normal conditions,NF-?B binds to NF-?B inhibitor?I?B?in the cytoplasm,and after activation,I?B protein induces the secretion of TNF-??interleukin-1??IL-1??and other proinflammatory cytokines,Some scholars have detected activated NF-?B in cardiac tissue after MI,and our team's previous work has shown that MI activates NF-?B and inhibits the activation of NF-?B down-regulate the expression of NGF,which can improve sympathetic remodeling.By observing changes in NF-?Bp65,purineergic signaling can activate NF-?B through the P2X-7 receptor.P2X₇R may be involved in the inflammatory process after cardiac ischemia,and potentially involved in regulation of NF-?B,However,whether P2X₇R is directlyinvolved in myocardial infarction after sympathetic remodeling still remains unknown.In this study,we hypothesized that the activation of P2X₇R was a key upstream factor in postmortematic neuronal remodeling.Firstly,we examined the dynamic expression of P2X₇R in the myocardium tissue of rats after MI,Secondly,we applied the targeted gene interference technique to silence the expression of P2X₇R expression,and further detected its role in the sympathetic nerve remodeling and its related mechanism.At the same time,we observed the effects of P2X₇R RNAi on cardiac function and ventricular arrhythmia,This study may provide a new theoretical basis and therapeutic target for the suppression of cardiac sympathetic remodeling,the reduction of malignant ventricular arrhythmias and sudden cardiac death in MI.In order to systematically explain the above problems,this study will be divided into the following two parts.Part 1.The expression of P2X₇ receptor in rates after myocardial infarctionObjectives:The model of myocardial infarction was established to determine whether P2X₇R was involved in the process of cardiac sympathetic remodeling after myocardial infarction.Methods:Twenty-seven SD rats were randomly divided into the following six groups:0-MI group,0.5-MI group,1-MI group,3-MI group,5-MI group and 7-MI group and blank control group?sham group?for each group.Permanent ligation of the left anterior descending coronary artery are used to establish the myocardial infarction model,Pericardiotomy without coronary artery ligation are used for sham model.0 day,1 day,3 days,5 days,7 days after MI the rats were sacrificed for detecting of P2X₇R by Western blots,Results:ECG monitoring showed ST elevation,suggesting that myocardial infarction model was successfuly established,Western blot analysis and RT-PCR showed that the expression of P2X₇R was slightly up-regulated at 0.5 day after myocardial infarction,and the expression was significantly up-regulated at the 1st day,It maintained at high level at day 7.Conclusions:Significant increase level of P2X₇R was observed in the infarcted lesion of rats after myocardial infarction,which was mildly expressed at 12 hours after infarct and maintained at a high level from the day 1 to the day 7,suggesting that P2X₇R is likely to regulate the expression of inflammatory factors after myocardial infarction,which may be involved in the regulation of sympathetic remodeling after infarction.Part 2.Effects and mechanisms of P2X₇ receptor on sympathetic remodeling and cardiac function in rats with myocardial infarctionObjectives:After injecting Ad-shRNA-P2X₇R into MI rats,the expression of P2X₇ receptor was observed to determine whether P2X₇ receptor was involved in the activation of NF-?B,the expression of inflammatory factor and the process of sympathetic remodeling.And to explore the therapeutic effect on the ventricular arrhythmia and improving cardiac function after myocardial infarction by finterfering with P2X₇ receptor.Methods:Forty-five SD rats were randomly divided into sham group?n = 15?,MI-vehicle group?MI+saline group?,MI-GFP group?MI+Ad-GFP-shRNA group?and MI-shRNA group?MI+Ad-P2X₇R-shRNA group??n=20?.After myocardial infarction was successfully constructed,the rats in each group were injected with the same amount solution?80.1?in the four parts of the infarct of the left ventricle??2mm in the infarct area?And the sham operation group didn't receive any rejection,MI-vehicle group received saline injection,MI-control group received Ad-GFP-shRNA injection,MI-shRNA group received Ad-P2X₇R-shRNA,On 7th day after infarction,echocardiographic experts were subjected to hemodynamic measurements and programmed electrical stimulation were used to induce arrhythmias for electrophysiological testing.Rats were then sacrificed and the myocardium were collected for the following tests.1.Real-time quantitative RT-PCR was used to detect the expression of P2X₇R mRNA in myocardium.2.The expression of P2X₇R,Akt,Ser473,ERK1/2,NF-?Bp65,NF-IkB? and NGF protein in myocardium were detected by Western blot.3.The expression of CD68 and IL-1? was observed by immunohistochemistry to understand the infiltration of inflammation.4.Masson staining on cardiac tissue sections assessed infarct size.5.Immunofluorescence was used to detect the silencing effect of P2X₇R gene.The morphological and distribution of TH and GAP43 were observed to understand the regeneration of sympathetic nerve.6.Enzyme-linked immunosorbent assay?ELISA?was used to detect the concentration of NE in myocardium to understand the sympathetic nerve activity.Results:1.Compared with sham group,LVEDP was significantly increased in MI groups,and EF,dP/dt max and dP/dt min were significantly decreased,and there was no significant difference between the two groups difference.Compared with MI-vehicle group LVEDP were significantly decreased in MI-shRNA group,and EF,dP/dt max and dP/dt min were significantly increased.2.The induced rates of ventricular arrhythmia in Sham group,MI-vehicle group,MI-GFP group and MI-shRNA group were 20%,62.5%,66.7%and 29.4%respectively.Compared with Sham group,susceptibility of arrhythmia in MI-vehicle group and MI-GFP group is higher.Compared with MI-vehicle group,the susceptibility of arrhythmia in MI-shRNA group was significantly lower than that in MI-vehicle group.3.The expression of P2X₇R in MI-vehicle group and MI-GFP group was significantly up-regulated compared with that of Sham.Compared with MI-vehicle group,the expression of P2XR in MI-shRNA group was significantly lower than that in MI-GFP group cut back.4.Compared with sham group,the expressions of NF-?B p65 were up-regulated in MI-vehicle group and MI-GFP group,while the expression of I?B? cytoplasm was decreased.The expression of NF-?B p65 in MI-shRNA group was decreased and the expression of NF-?B was increased in MI-shRNA group compared with MI-GFP group,and the expression of NF-?B p65 in cytoplasmic protein increased.The activation NF-?B signaling pathwaywas significantly inhibited.5.Compared with sham group,the expression of CD68 and IL-1? in MI groups were significantly increased,and there was no significant difference between MI-GFP group and MI-GFP group.The expression of CD68 and IL-1? in MI-shRNA group was significantly decreased,which indicated that the inflammatory infiltration was obviously weakened.6.Compared with the sham group,the expression of NGF protein in MI groups was significantly higher than that in MI-sham group.Compared with MI-vehicle group,the expression of NGF protein in MI-shRNA group was significantly decreased.7.?1?GAP43 positive nerve fibers:GAP43 positive nerve fibers in Sham group were rare or even absent;compared with Sham group,the density of GAP43 positive nerve fibers in MI groups was significantly higher than that in MI-sham group The density of nerve fibers in MI-shRNA group was significantly decreased.?2?TH-positive nerve fibers:Sham group TH-positive nerve fibers were evenly distributed in the myocardial fibers,and keep the same shape with the myocardial fiber;Compared with the Sham group,the density of TH-positive nerve fibers in MI groups were significantly higher than that in MI-sham group,and there was no significant difference between the two groups.Compared with MI-vehicle group,The density of TH-positive nerve fibers in MI-shRNA group was significantly decreased,and the morphology and distribution were almost normal.8.Compared with sham group,the level of NE of myocardium in MI-vehicle group and MI-GFP group was significantly higher.It is lower than that in MI-vehicle group after MI P2X₇R silencing,the sympathetic nerve activity was significantly increased in MI-vehicle group Group and MI-GFP group and decreased after P2X₇R silence.Conclusions:1.P2X₇R up-regulated in MI,then NF-?B signaling pathway was activated,macrophage infiltration increased,IL-1? up-regulated,NGF expression,sympathetic remodelingup-regulated,ventricular arrhythmia susceptibility increased;Heart function deteriorated.2.The expression of P2X₇R gene was successfully silenced by targeted injection of Ad-P2X₇R-shRNA.3.The expression of P2X₇R was inhibited by short hairpin RNA,which could inhibit the activation of NF-?B signaling pathway,decrease the expression of IL-1?,and down-regulate the expression of NGF.4.P2X₇R gene silencing can ameliorate the sympathetic nerve regeneration after MI to play the role of antiarrhythmic and improving heart function.