| Background and ObjectiveCardiovascular disease is the leading cause of death worldwide.Myocardial infarction(MI)is myocardial necrosis caused by acute and persistent coronary ischemia and hypoxia and may lead to myocardial injury,cardiogenic shock,and cardiac arrest.Ventricular arrhythmia(VA)is a common complication of MI,while primary arrhythmia is the main cause of sudden cardiac death in patients with MI.Although anti-arrhythmic drugs exhibit cardioprotective effects,most of them have poor curative efficiency and a relatively high risk of adverse reactions.Therefore,finding new therapeutic targets and exploring the underlying mechanism of arrhythmia is of great significance for reducing mortality after MI.Recent studies have shown that the occurrence of arrhythmia is closely related to the electrical coupling disorder of cardiomyocytes.A gap junction is a transmembrane channel for electrical and chemical coupling between cardiomyocytes.Connexin(Cx)is essential for maintaining communication connections between cardiomyocytes,electrical conduction,and normal rhythmic contraction.Gap junction channels form an intercellular pathway for electrical coupling between cells,which is essential for the transmission of normal heart impulses.Cx43 is the main connexin of ventricular muscle gap junction channels.Abnormal changes in its quantity,distribution,and phosphorylation level will cause the overall abnormality of cardiomyocytes,change the conduction velocity and direction,produce myocardial reentry,conduction block,and cause arrhythmia.MI is closely related to aseptic inflammation and plays a crutial role in the tissue healing process.However,excessive inflammation can lead to heart damage and ventricular remodeling.Interleukin-1β(IL-1β)is a key inflammatory factor that regulates heart inflammation and repair.Recent studies have shown that IL-1β is closely related to the occurrence of arrhythmia,and treatment of IL-1β in vitro can reduce the expression of Cx43 in astrocytes and cardiomyocytes.NLRP3 inflammasome is currently the most studied inflammasome,which is composed of NLRP3,ASC and pro-caspase-1.After being stimulated by pathogenic toxins or danger signals,the NLRP3 protein will recruit ASC and pro-caspase-1,resulting in the cleavage of pro-caspase-1 into active caspase-1,and caspase-1 will hydrolyze the pro-IL-1β protein Mature and active IL-1β.Therefore,we speculated that the caspase-1/IL-1β pathway may participate in the change of Cx43 expression after MI and affect the communication between cells.Based on the above speculation,this project intends to establish a rat MI model.The rat primary fibroblasts(RCFs)and rat primary cardiomyocytes(RCMs)are used to develop a cellular inflammation model in vitro,by using the caspase-1 inhibitor VX765 pretreatment method,followed by observing the effect of VX765 on Cx43 expression and intercellular communication after MI and to clarify the molecular mechanism of caspase-1/IL-1β pathway regulating Cx43,providing a new therapeutic target for myocardial injury after MI.Methods1.The effect of VX765 on the heart function of rats after MIThe rat MI model was established by ligating the left anterior descending coronary artery,and the male Sprague-Dawley(SD)rats were divided into three groups,the sham group(only threading but not ligation),and the operation group(MI)and the administration group(MI/VX765)(VX765 was injected into the tail vein at16 mg/kg 1 h before the operation,for 7 days,once a day).HE staining,Masson staining and TTC staining were used to detect the effects of VX765 on cardiac tissue morphology,fibrosis and infarct area in MI rats.Electrocardiogram,echocardiogram and serum myocardial enzymes were used to detect the effects of VX765 on cardiac function and QT interval in rats after MI.The enzyme linked immunosorbent assay(ELISA)was used to determine serum IL-1β and LDH.2.The effect of VX765 on NLRP3/caspase-1/IL-1β pathway and Cx43 after MI in ratsImmunohistochemical method was used to detect the expression of caspase-1,IL-1β and Cx43 in heart tissue.Western bolt method was used to detect the expression changes of NLRP3,ASC,caspase-1,IL-1β and Cx43 protein levels in rat MI tissues.3.The effect of VX765 on NLRP3/caspase-1/IL-1β pathway in RCFsEnzymatic digestion method was used to extract RCFs and RCMs to establish RCFs inflammation model.VX765(25 μmmol/L)was pretreated for 30 minutes,LPS(1 μg/m L)was incubated for 12 h and ATP(5 mmol/L)was incubated for 1 h,and then NLRP3,ASC,caspase-1 and IL-1β protein expression changes in RCFs were detected by Western bolt method,and ELISA was used to determine the changes in IL-1β content in the cell supernatant.4.The effect of VX765 inhibiting the secretion of IL-1β in RCFs on the expression of Cx43 in RCMsTo prove whether IL-1β secreted by NLRP3 inflammasome activation in RCFs can regulate Cx43 of RCMs by paracrine mode.The supernatant of LPS/ATP-stimulated RCFs was used as the conditioned medium to incubate RCMs for 24 h.Western bolt method was used to detect the expression of Cx43 protein in RCMs.Scratch dye transfer technology and immunofluorescence staining were used to evaluate the changes in communication connection function and the localization of Cx43 in RCMs.5.The molecular mechanism of the effect of caspase-1/IL-1β on Cx43 in RCMsIt is reported that IL-1β induces p38 MAPK activation in astrocytes and inhibits Cx43-mediated dye coupling of gap junctions.We used Western bolt method to detect the activation of p38 MAPK in RCMs and rat heart tissues.After treatment with IL-1β receptor antagonist p-38 MAPK inhibitor SB203580(3 μmol/L)or TLR1(40μmol/L)for 1 h,RCMs were incubated with recombinant IL-1β(80 ng/m L)for 24 h.Western bolt method was used to detect the expression changes of Cx43 and p38 MAPK protein levels in RCMs.Scratch dye transfer technology and immunofluorescence staining were used to detect changes in the communication function between RCMs and changes in Cx43 localization.Results1.VX765 can alleviate the changes in cardiac morphology and cardiac dysfunction after MI in ratsCardiac cells in MI group had varying degrees of vacuolization and inflammatory infiltration,increased fibrosis,and increased myocardial infarction area,while MI/VX765 group could improve the occurrence of cell malignancy and reduce the degree of cardiac fibrosis,leading to the area of myocardial infarction decreased from 41.58 ± 5.21% to 15.07±2.52%(P < 0.05).Compared with the sham group,LVEF decreased from 69.30±7.62% to 44.38±7.76%(P < 0.01),left ventricular fractional shortening(LVFS)decreased from 46.75±5.88% to 23.53±1.84%(P <0.01),left ventricular diastole.The left ventricular end-diastolic dimension(LVED.d)increased from 4.63±0.35% to 7.63±0.47%(P < 0.01),suggesting that cardiac dysfunction occurred,and the cardiac function of MI/VX765 group recovered significantly(P < 0.01).In addition,the level of LDH in the MI group increased to1330.00±153.30 U/L(P < 0.001),while VX765 pretreatment reduced its level(598.60±143.10 U/L,P < 0.01).The electrocardiogram results showed that the ST segment elevation in the MI group prolonged the QT interval and QTc interval,while VX765 treatment could reduce the ST segment elevation amplitude and significantly shorten the QT interval and QTc interval(P < 0.01).2.VX765 can inhibition the activation of NLRP3/caspase-1/IL-1β pathway after MI in ratsThe results of immunohistochemistry showed that the expression of caspase-1and IL-1β in the infarct zone and border zone of the MI group increased significantly,but decreased significantly after pretreatment with VX765(P < 0.05).In addition,the protein components(NLRP3,ASC,caspase-1)and IL-1β of NLRP3 inflammasome in MI group increased significantly(P < 0.05);IL-1β in serum increased from588.10±162.80 pg/m L to 4723.00±666.70 pg/m L(P <0.001),while pretreatment with VX765 reduced these protein levels(P < 0.05)and serum IL-1β secretion levels.3.VX765 can increase the expression of Cx43 protein and improve its distribution after MI in ratsIn normal heart tissue,Cx43 is distributed in clusters,mainly at the end-to-end junctions of adjacent cells,and a small amount at the cell side junctions.In the MI group,Cx43 was almost invisible around the infarct focus,rarely distributed at the cell side junction or scattered on the cell surface,and the protein expression level was significantly reduced(P < 0.01).After VX765 pretreatment,this abnormal distribution was significantly improved,and the expression of Cx43 protein was significantly increased(P <0.01).4.VX765 can inhibition the activation of NLRP3/caspase-1/IL-1β pathway in RCFsAfter LPS/ATP stimulated RCFs,NLRP3,ASC,caspase-1,and IL-1β proteins were significantly increased(P < 0.05),and the IL-1β content in the cell supernatant rose from 52.98±7.64 pg/m L to 902.70±23.28 pg/m L(P < 0.01),and VX765 pretreatment can significantly reduce the increase of these proteins and the secretion of IL-1β in the cell supernatant(P < 0.01).5.The activation of caspase-1/IL-1β pathway in RCFs participates in the regulation of Cx43 in RCMsAfter LPS/ATP stimulated RCFs supernatants were incubated in cardiomyocytes,Cx43 protein expression in RCMs significantly decreased(P < 0.05),the transfer area of fluorescein yellow decreased(P < 0.01),and the expression of Cx43 in the membrane decreased and the expression increased in the cytoplasm.VX765 pretreatment increased the expression of Cx43 protein(P < 0.01)and improved intercellular communication function and internalization of Cx43.6.Caspase-1/IL-1β may regulate the expression of Cx43 in RCMs cells through the p38 MAPK pathwayWe first observed that the ratio of p-p38 MAPK/p38 MAPK increased in MI rat heart tissue and RCMs incubated with the supernatant of LPS/ATP-stimulated RCFs cells(P < 0.01),while VX765 pretreatment could decrease the ratio of p-p38MAPK/p38 MAPK.(P < 0.01).To further observe the direct effect of IL-1β on Cx43,after incubating RCMs with exogenous IL-1β,the expression of Cx43 protein was significantly reduced(P < 0.05),p-p38 MAPK protein was significantly increased(P< 0.05),the transfer area of fluorescein yellow decreases,and the internalization of Cx43 increases.After pretreatment with p-38 MAPK inhibitor SB203580 and IL-1βreceptor antagonist,Cx43 protein expression increased significantly(P < 0.05),fluorescein yellow transfer increased(P < 0.05),and internalization decreased.ConclusionCaspase-1 inhibitor VX765 can up-regulate the expression of Cx43 after myocardial infarction in rats and improve the communication function between cells. |
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