| Objective:Myocardial ischemia reperfusion(I/R)injury is a major challenge in the treatment of acute myocardial infarction.The gap junction formed by connexin 43(Cx43)can promote the rapid propagation of action potentials and coordinate the contraction of the heart,which is essential for the development and function of the heart.When myocardial I/R injury occurs,gap junction Cx43 is degraded by ubiquitination,resulting in increased Cx43 hemichannel activity.In addition,the abnormally activated Cx43 hemichannel is related to the inflammatory damage mediated by pyroptosis.The present study aimed to investigate the roles and mechanisms of ubiquitin-specific peptidase 8(USP8),Cx43,its hemichannels and pyroptosis in myocardial I/R injury.It provides a new target and pathway for the prevention and treatment of ischemic heart disease.Methods:1.The myocardial I/R injury model was established by ligating the anterior descending branch of the left coronary artery for 30 minutes and reperfusion for 2 hours.TTC staining was used to detect myocardial infarction size.2.Myocardial structural changes were observed by HE staining.The serum levels of CK-MB,c Tn I and IL-1β were determined by ELISA kit.3.Tissue immunofluorescence assay was used to observe the phenomenon of myocardial intercalated disk Cx43 remodeling.The expression level of NLRP3 inflammasome in myocardium was observed by immunohistochemical staining.4.H9c2 cells were hypoxic for 6 h and reoxygenated for 12 h to established hypoxia reoxygenated(H/R)injury model.Morphological changes of cells were observed under microscope.The proportion of PI positive cells was assessed by Hoechst 33342/PI double staining assay.LDH release rate was detected by LDH detection kit.5.The Cx43 gap junction intercellular communication(GJIC)function was evaluated with the Calcein AM/Di I double staining assay.Et Br dye uptake assay was used to assess the activity of Cx43 hemichannel,and ATP release was detected with ATP detection kit.6.The overexpressed USP8 plasmid was constructed by seamless cloning method and identified by agarose gel electrophoresis and sequencing results.7.The expression levels of USP8,NLRP3,P2X7,Cx43,GSDMD-N,ASC,cleaved caspase-1 and cleaved IL-1β in rat myocardial tissue and H9c2 cells were determined by Western blot.Results:1.Severe myocardial injury was induced by I/R treatment in rats.Compared with sham group,myocardial infarction area was significantly increased,myocardial tissue structure disorder,the levels of CK-MB,c Tn I and IL-1β in serum were apparently elevated,and the expressions of pyroptosis-related proteins(NLRP3,GSDMD-N,ASC,cleaved-caspase-1 and cleaved-IL-1β)were up-regulated in I/R group.2.H/R injury induced pyroptosis of H9c2 cells.Compared with control group,H9c2 cells extended large pyrolytic bubbles from the plasma membrane,the expression levels of pyroptosis-related proteins were up-regulated,the PI positive rate and the LDH release rate elevated notably in H/R group.3.Inhibition of P2X7 receptor alleviated H/R-induced pyroptosis of H9c2 cells.H9c2 cells were pretreated with 10 μM P2X7 receptor inhibitor(A-740003)for 1 h,followed by H/R treatment.Compared with H/R group,the expressions of NLRP3 inflammasome and pyroptosis-related proteins were effectively reduced,as well as the PI positive rate and the LDH release rate in A-740003+H/R group.4.Cx43 was involved in myocardial I/R injury.Compared with sham group,the expression of Cx43 in myocardial tissue was down-regulated,and the Cx43 in intercalated disk was reconstructed laterally in I/R group.Compared with control group,the expression of Cx43 in H9c2 cells was down-regulated,the GJIC function was weakened,while the activity of hemichannel was enhanced,and the release rate of ATP was significantly increased in H/R group.5.Inhibition of Cx43 hemichannel activity alleviated pyroptosis and myocardial I/R injury.Cx43 hemichannel inhibitor(Gap19)was injected into caudal vein at a dose of 12.5 mg/kg 10 min before surgery,followed by rat myocardial I/R experiment.Compared with I/R group,myocardial infarction area,the CK-MB,c Tn I and IL-1βlevels in serum were obviously decreased,myocardial tissue structure was effectively improved,the expressions of NLRP3 inflammasome,P2X7 and pyroptosis-related proteins were down-regulated in Gap19+I/R group.6.Inhibition of Cx43 hemichannel activity alleviated P2X7/NLRP3 mediated pyroptosis.H9c2 cells were pretreated with 250 μM Gap19 inhibitor for 30 min,followed by H/R treatment.Compared with H/R group,the activity of Cx43 hemichannel was inhibited,the ATP release rate was notably reduced,the expressions of P2X7 and pyroptosis-related proteins were declined,as well as the PI positive rate and the LDH release rate in Gap19+H/R group.7.Overexpression of Cx43 inhibited its hemichannel activity and alleviated P2X7/NLRP3 mediated pyroptosis.Compared with GV-NC+H/R group,Cx43 protein expression was up-regulated,hemichannel activity was decreased,ATP release was markedly weakened,the expressions of P2X7 and pyroptosis-related proteins were reduced,as well as the PI positive rate and the LDH release rate in GV-Cx43+H/R group.8.Overexpression of USP8 inhibited P2X7/Nl RP3 mediated pyroptosis by stabilizing Cx43 and regulating its hemichannel activity.Compared with pc-NC+H/R group,the protein expressions of USP8 and Cx43 were effectively saved,the GJIC function was improved,the activity of Cx43 hemichannel was decreased,ATP release was effectively declined,the expressions of P2X7 and pyroptosis-related proteins were reduced,as well as the PI positive rate and the LDH release rate in pc-USP8+H/R group.Conclusions:Deubiquitinase USP8 stabilizes Cx43 and regulates its hemichannel activity,inhibits P2X7/Nl RP3-mediated pyroptosis pathway,and thus alleviates myocardial I/R injury. |
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