IP3R1调控CaMKⅡ和VDAC1在海洛因致心肌细胞节律异常中的作用

Role of IP3R1 in regulation of CaMKII and VDAC1 in heroin-induced cardiomyocyte rhythm abnormalities

目的:探讨1,4,5-三磷酸肌醇1型受体(inositol 1,4,5-trisphosphate receptor type 1,IP3R1)调控钙/钙调蛋白依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)和电压依赖性阴离子通道1(voltage-dependent anion channel 1,VDAC1)在海洛因(heroin,HE)致心肌细胞节律异常中的作用。方法:联合蛋白组学和GEO(Gene Expression Omnibus)数据库分析心律失常芯片数据,寻找关键调控因子。构建IP3R1基因敲减慢病毒并感染原代乳大鼠心肌细胞(neonatal rat cardiomyocytes,NRCMs),实验分为对照(control)组、HE组和HE+shIP3R1组。结晶紫染色观察心肌细胞形态;ELISA法检测乳酸脱氢酶(lactate dehydrogenase,LDH)和天冬氨酸转氨酶(aspartate aminotransferase,AST)水平;透射电镜观察线粒体形态学变化;Fluo-4/AM探针法检测细胞内Ca^(2+)浓度;DCFH-DA荧光探针检测细胞内活性氧(reactive oxygen species,ROS)含量;JC-1染色法检测线粒体膜电位(mitochondrial membrane potential,MMP)水平;ATP检测试剂盒检测细胞内ATP水平;免疫共沉淀(co-immunopre-cipitation,Co-IP)分析IP3R1与CaMKⅡδ和VDAC1蛋白之间的相互作用;Western blot检测IP3R1、CaMKⅡδ、p-CaM-KⅡδ(T287)和VDAC1的蛋白水平。结果:结合蛋白质组学和基因表达谱数据集GSE89410分析,筛选得到80个差异共表达分子,基于基因本体论(Gene Ontology,GO)功能注释和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析结果,最终筛选出关键因子IP3R1,且通过STRING数据库获得IP3R1结合蛋白:CaMKⅡδ和VDAC1。Co-IP结果验证IP3R1与CaMKⅡδ和VDAC1存在相互作用,且HE干预后NRCMs中IP3R1与CaMKⅡδ和VDAC1之间的相互作用增强。体外细胞实验显示,与control组相比,HE组NRCMs数量急剧减少,细胞膜变窄,伪足减少,细胞核结构模糊;LDH和AST水平均显著上升(P<0.05);线粒体超微结构损伤严重,证实HE对NRCMs具有毒性作用并导致线粒体损伤。与control组相比,HE组心肌细胞内Ca^(2+)浓度、ROS水平、MMP以及IP3R1、p-CaMKⅡδ(T287)和VDAC1蛋白水平均显著升高(P<0.05),而HE+shIP3R1组这些指标均显著减低(P<0.05);ATP水平则相反。这证实沉默IP3R1表达可减轻HE干预后NRCMs的钙超载及线粒体损伤。结论:IP3R1通过调控CaMKⅡ和VDAC1引起心肌细胞钙超载和ROS生成增多,参与HE诱导的心肌细胞节律异常。

AIM:To investigate the role of inositol 1,4,5-trisphosphate receptor type 1(IP3R1)in the regulation of calcium/calmodulin-dependent protein kinase II(CaMKII)and voltage-dependent anion channel 1(VDAC1)in heroin(HE)-induced cardiomyocyte rhythm abnormalities.METHODS:In this study,we analysed arrhythmia microarray data by combining proteomics and Gene Expression Omnibus(GEO)to search for key regulators.Lentiviral vector with knockdown of IP3R1 gene was constructed,and used to infect neonatal rat cardiomyocytes(NRCMs).The cells were divided into control group,HE group,and HE+shIP3R1 group.Crystal violet staining was used to observe the morphology of cardiomyocytes.Lactate dehydrogenase(LDH)and aspartate aminotransferase(AST)levels were detected by ELISA.Transmission electron microscopy was used to observe the morphological changes of mitochondria.Intracellular Ca^(2+)concentration was detected by Fluo-4/AM probe method.Intracellular reactive oxygen species(ROS)content was detected by DCFH-DA fluorescent probe.Mitochondrial membrane potential(MMP)level was detected by JC-1 staining method.Intracellular ATP level was detected by ATP assay kit.The interaction between IP3R1 and CaMKIIδ/VDAC1 was verified by co-immunoprecipitation(Co-IP)detection.The protein levels of IP3R1,CaMKIIδand p-CaMKIIδ(T287)and VDAC1 were detected by Western blot.RESULTS:Combined with proteomics and gene expression profiling dataset GSE89410 analysis,80 differentially co-expressed molecules were screened.Based on the results of Gene Ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis,the key factor IP3R1 was finally screened out,and IP3R1-binding proteins were obtained from the STRING database:CaMKIIδand VDAC1.Moreover,the Co-IP results verified the interaction between IP3R1 and CaMKIIδ/VDAC1,and the interaction between IP3R1 and CaMKIIδ/VDAC1 was increased in the NRCMs after HE intervention.In vitro cellular experiments showed that compared with control group,the number of NRCMs in HE group was drastically reduced,the cell membrane was narrowed,the pseudopods were reduced,and the structure of the nucleus was blurred.The levels of both LDH and AST were significantly increased(P<0.05),and the mitochondrial ultrastructural damage was severe.It was confirmed that HE enhanced the cardiotoxic effects and mitochondrial damage in NRCMs.Compared with control group,intracardiomyocyte Ca^(2+)concentration,ROS levels,MMP,and IP3R1,p-CaMKIIδ(T287)and VDAC1 protein levels were significantly elevated in HE group(P<0.05)and significantly attenuated in HE+shIP3R1 group(P<0.05),but the ATP level was the opposite.It was confirmed that silencing of IP3R1 attenuated cellular calcium overload and mitochondrial damage in NRCMs after HE intervention.CONCLUSION:The IP3R1 is involved in HE-induced cardiomyocyte rhythm abnormality by regulating CaMKII and VDAC1 to cause cardiomyocyte calcium overload and increased ROS production.