Background:Abnormal myocardial voltage-gated sodium channel 1.5(Nav1.5)expression and function cause lethal ventricular arrhythmias during myocardial ischemia–reperfusion(I/R).Protein inhibitor of activated STAT Y(PI...Background:Abnormal myocardial voltage-gated sodium channel 1.5(Nav1.5)expression and function cause lethal ventricular arrhythmias during myocardial ischemia–reperfusion(I/R).Protein inhibitor of activated STAT Y(PIASy)-mediated caveolin-3(Cav-3)small ubiquitin-related modifier(SUMO)modification affects Cav-3 binding to the Nav1.5.PIASy activity is increased after myocardial I/R,but it is unclear whether this is attributable to plasma membrane Nav1.5 downregulation and ventricular arrhythmias.Methods:Using recombinant adeno-associated virus subtype 9(AAV9),rat cardiac PIASy was silenced using intraventricular injection of PIASy short hairpin RNA(shRNA).After two weeks,rat hearts were subjected to I/R and electrocardiography was performed to assess malignant arrhythmias.Tissues from peri-infarct areas of the left ventricle were collected for molecular biological measurements.Results:PIASy was upregulated by I/R(P<0.01),with increased SUMO2/3 modification of Cav-3 and reduced membrane Nav1.5 density(P<0.01).AAV9-PIASy shRNA intraventricular injection into the rat heart down-regulated PIASy after I/R,at both mRNA and protein levels(P<0.05 vs.Scramble-shRNA+I/R group),decreased SUMO-modified Cav-3 levels,enhanced Cav-3 binding to Nav1.5,and prevented I/R-induced decrease of Nav1.5 and Cav-3co-localization in the intercalated disc and lateral membrane.PIASy silencing in rat hearts reduced I/R-induced fatal arrhythmias,which was reflected by a modest decrease in the duration of ventricular fibrillation(VF;P<0.05 vs.Scramble-shRNA+I/R group)and a significantly reduced arrhythmia score(P<0.01 vs.Scramble-shRNA+I/R group).The anti-arrhythmic effects of PIASy silencing were also evidenced by decreased episodes of ventricular tachycardia(VT),sustained VT and VF,especially at the time 5–10 min after ischemia(P<0.05 vs.Scramble-shRNA+IR group).Using in vitro human embryonic kidney 293 T(HEK293T)cells and isolated adult rat cardiomyocyte models exposed to hypoxia/reoxygenation(H/R),we confirmed that increased PIASy promoted Cav-3 modification by SUMO2/3 and Nav1.5/Cav-3 dissociation after H/R.Mutation of SUMO consensus lysine sites in Cav-3(K38R or K144R)altered the membrane expression levels of Nav1.5 and Cav-3 before and after H/R in HEK293T cells.Conclusions:I/R-induced cardiac PIASy activation increased Cav-3 SUMOylation by SUMO2/3 and dysregulated Nav1.5-related ventricular arrhythmias.Cardiac-targeted PIASy silencing mediated Cav-3 deSUMOylation and partially prevented I/R-induced Nav1.5 downregulation in the plasma membrane of cardiomyocytes,and subsequent ventricular arrhythmias in rats.PIASy was identified as a potential therapeutic target for life-threatening arrhythmias in patients with ischemic heart diseases.展开更多
目的:探讨磁性Fe_3O_4纳米颗粒对大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain蛋白表达的影响,阐明其作用机制。方法:将24只Wistar大鼠按体质量随机分成对照组和低、中、高剂量磁性Fe_3O_4纳米颗粒组,尾静脉注射不同剂量磁性Fe_...目的:探讨磁性Fe_3O_4纳米颗粒对大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain蛋白表达的影响,阐明其作用机制。方法:将24只Wistar大鼠按体质量随机分成对照组和低、中、高剂量磁性Fe_3O_4纳米颗粒组,尾静脉注射不同剂量磁性Fe_3O_4纳米颗粒24h后取脏器组织,Western blotting法检测大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain蛋白的表达水平,荧光实时定量PCR法检测大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain mRNA的表达水平。结果:与对照组比较,中和高剂量组大鼠肝脏和脾脏组织中Clathrin Heavy Chain蛋白和mRNA表达水平明显升高(P<0.05)。高剂量组大鼠肾脏组织中Clathrin Heavy Chain mRNA的表达水平与其他3组比较明显升高(P<0.05)。Caveolin-1蛋白表达水平在各剂量组之间比较差异无统计学意义(P>0.05);与对照组比较,低、中和高剂量组大鼠肝脏、肺脏和脾脏组织中Caveolin-1mRNA表达水平明显升高(P<0.05);各组肾脏组织中Caveolin-1 mRNA表达水平差异无统计学意义(P>0.05)。结论:磁性Fe_3O_4纳米颗粒能够诱导大鼠肝脏、肺脏、脾脏中Clathrin Heavy Chain蛋白表达增强,通过Clathrin Heavy Chain蛋白的内吞作用是磁性Fe_3O_4纳米颗粒进入大鼠肝脏、肺脏和脾脏细胞的途径之一。展开更多
Objective:To discuss the abnormal expression of Wnt inhibitory factor(WIFI) in hepatocellular carcinoma cells and its regulating effect on the hepatocellular carcinoma invasion and metastasis factors of tissue inhibit...Objective:To discuss the abnormal expression of Wnt inhibitory factor(WIFI) in hepatocellular carcinoma cells and its regulating effect on the hepatocellular carcinoma invasion and metastasis factors of tissue inhibitor of matrix metalloproteinases-3(TIMP-3)and caveolin-1.Methods:RT-PCR and Western blot were employed to detect the expression of WIF1 in six hepatocellular carcinoma eell lines of HepG2,Hep3 B,Huh7,PLC/PRF/5.SMMC-7721 and MHCC97 and the immortalized human liver cell line THLE-3.Besides,Lipofectamine 2000 was employed to transfect the eukaryotic expression vector pcDNA3.lWIF1 and blank plasmid pcDNA3.1 into hepatocellular carcinoma cell lines.Transwell assay was used to detect the effect of WIF1 on the invasion ability of hepatocellular carcinoma cells;Western blot was used to detect the effect of WIF1 on the expression of TIMP-3 and caveolin-1in hepatocellular carcinoma cells,it also discussed the effect on the expression of β-catenin.Results:The expression of WIF1 in hepatocellular carcinoma cell lines was lower than that in the normal liver cell lines(P<0.01);while there was basically no expression of WIF1 in the human highly metastatic cell line MHCC-97 and moderate expression in HepG2 and SMMC-7721.Therefore,HepG2 and SMMC-7721 were chosen as the further experimental cell lines.After transfecting the eukaryotic expression vector peDNA3.1-WEFl and blank plasmid pcDNA3.1 into hepatocellular carcinoma eell lines,compared with(he blank plasmid group,the cell viability and invasion ability in the WIF1 group were all reduced(P<0.01),the expression of TIMP-3,caveolin-1 and mRNA were all down-regulated(P<0.01),and the expression of β-catenin was decreased(P<0.01).Conclusions:Because of down-regulation or missing of expression of WIFI in hepatocellular carcinoma cell lines,the up-regulation of WIFI expression can significantly inhibit the invasion and metastasis of HepG2 and SMMC-7721 of hepatocellular carcinoma cell lines,which are related to the up-regulated expression of TIMP-3 and down-regulated expression of caveolin-1 and may be realized through the Wnt/β-catenin signaling pathway.展开更多
Objective:To observe whether CAV3 is involved in the physiological process of IMGU and NIMGU.Down-regulation of caveolin-3(CAV3)protein expression by small interference RNAs(iRNA)has been studied.Insulin-mediated gluc...Objective:To observe whether CAV3 is involved in the physiological process of IMGU and NIMGU.Down-regulation of caveolin-3(CAV3)protein expression by small interference RNAs(iRNA)has been studied.Insulin-mediated glucose uptake(IMGU)is critical in skeletal muscle and cardiac myocytes,but non-insulin-mediated glucose uptake(NIMGU)should not be neglected.Methods:CAV3 siRNAs were designed and transfected in C2C12 cells and H9c2 cells in skeletal muscle and cardiac muscle,respectively,and C2C12 and H9c2 cells were cultured in DMEM medium with and without insulin,respectively.Glucose transporter 4(GLUT4)protein expression was detected by Western blot,and the glucose uptake rate of cells was measured by biochemical kit.Results:Transfection with CAV3 siRNA successfully down-regulated CAV3 protein expression in C2C12 and H9c2 cells.In the absence of insulin stimulation,GLUT4 expression was decreased(P<0.01)and glucose uptake was reduced(P<0.05)after 48 h of transfection in C2C12 cells,and GLUT4 expression was decreased(P<0.05)and glucose uptake was reduced(P<0.01)after 48 h of transfection in H9c2 cells.In the presence of insulin stimulation,GLUT4 expression was decreased(P<0.01)and glucose uptake was reduced(P<0.01)after 48 h of transfection in C2C12 cells,and the downregulation of GLUT4 was not statistically significant and glucose uptake was reduced(P<0.01)after 48 hours of transfection in H9c2 cells.Conclusion:Two different states,IMGU and NIMGU,exist in C2C12 cells and H9c2 cells.Both in the quiet state stimulated by insulin as well as in the absence of insulin stimulation,the cellular uptake of glucose is affected by GLUT4 changes regulated by CAV3.展开更多
基金supported by grants from the National Natural Science Foundation of China(81770824,81270239)。
文摘Background:Abnormal myocardial voltage-gated sodium channel 1.5(Nav1.5)expression and function cause lethal ventricular arrhythmias during myocardial ischemia–reperfusion(I/R).Protein inhibitor of activated STAT Y(PIASy)-mediated caveolin-3(Cav-3)small ubiquitin-related modifier(SUMO)modification affects Cav-3 binding to the Nav1.5.PIASy activity is increased after myocardial I/R,but it is unclear whether this is attributable to plasma membrane Nav1.5 downregulation and ventricular arrhythmias.Methods:Using recombinant adeno-associated virus subtype 9(AAV9),rat cardiac PIASy was silenced using intraventricular injection of PIASy short hairpin RNA(shRNA).After two weeks,rat hearts were subjected to I/R and electrocardiography was performed to assess malignant arrhythmias.Tissues from peri-infarct areas of the left ventricle were collected for molecular biological measurements.Results:PIASy was upregulated by I/R(P<0.01),with increased SUMO2/3 modification of Cav-3 and reduced membrane Nav1.5 density(P<0.01).AAV9-PIASy shRNA intraventricular injection into the rat heart down-regulated PIASy after I/R,at both mRNA and protein levels(P<0.05 vs.Scramble-shRNA+I/R group),decreased SUMO-modified Cav-3 levels,enhanced Cav-3 binding to Nav1.5,and prevented I/R-induced decrease of Nav1.5 and Cav-3co-localization in the intercalated disc and lateral membrane.PIASy silencing in rat hearts reduced I/R-induced fatal arrhythmias,which was reflected by a modest decrease in the duration of ventricular fibrillation(VF;P<0.05 vs.Scramble-shRNA+I/R group)and a significantly reduced arrhythmia score(P<0.01 vs.Scramble-shRNA+I/R group).The anti-arrhythmic effects of PIASy silencing were also evidenced by decreased episodes of ventricular tachycardia(VT),sustained VT and VF,especially at the time 5–10 min after ischemia(P<0.05 vs.Scramble-shRNA+IR group).Using in vitro human embryonic kidney 293 T(HEK293T)cells and isolated adult rat cardiomyocyte models exposed to hypoxia/reoxygenation(H/R),we confirmed that increased PIASy promoted Cav-3 modification by SUMO2/3 and Nav1.5/Cav-3 dissociation after H/R.Mutation of SUMO consensus lysine sites in Cav-3(K38R or K144R)altered the membrane expression levels of Nav1.5 and Cav-3 before and after H/R in HEK293T cells.Conclusions:I/R-induced cardiac PIASy activation increased Cav-3 SUMOylation by SUMO2/3 and dysregulated Nav1.5-related ventricular arrhythmias.Cardiac-targeted PIASy silencing mediated Cav-3 deSUMOylation and partially prevented I/R-induced Nav1.5 downregulation in the plasma membrane of cardiomyocytes,and subsequent ventricular arrhythmias in rats.PIASy was identified as a potential therapeutic target for life-threatening arrhythmias in patients with ischemic heart diseases.
文摘目的:探讨磁性Fe_3O_4纳米颗粒对大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain蛋白表达的影响,阐明其作用机制。方法:将24只Wistar大鼠按体质量随机分成对照组和低、中、高剂量磁性Fe_3O_4纳米颗粒组,尾静脉注射不同剂量磁性Fe_3O_4纳米颗粒24h后取脏器组织,Western blotting法检测大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain蛋白的表达水平,荧光实时定量PCR法检测大鼠主要脏器组织中Caveolin-1及Clathrin Heavy Chain mRNA的表达水平。结果:与对照组比较,中和高剂量组大鼠肝脏和脾脏组织中Clathrin Heavy Chain蛋白和mRNA表达水平明显升高(P<0.05)。高剂量组大鼠肾脏组织中Clathrin Heavy Chain mRNA的表达水平与其他3组比较明显升高(P<0.05)。Caveolin-1蛋白表达水平在各剂量组之间比较差异无统计学意义(P>0.05);与对照组比较,低、中和高剂量组大鼠肝脏、肺脏和脾脏组织中Caveolin-1mRNA表达水平明显升高(P<0.05);各组肾脏组织中Caveolin-1 mRNA表达水平差异无统计学意义(P>0.05)。结论:磁性Fe_3O_4纳米颗粒能够诱导大鼠肝脏、肺脏、脾脏中Clathrin Heavy Chain蛋白表达增强,通过Clathrin Heavy Chain蛋白的内吞作用是磁性Fe_3O_4纳米颗粒进入大鼠肝脏、肺脏和脾脏细胞的途径之一。
基金supported by National Science&Technology Pillar Program during the 12th Five-year Plan Period under the research and development of new trisacryl gelatin microspheres(No.2012BAH5B06)
文摘Objective:To discuss the abnormal expression of Wnt inhibitory factor(WIFI) in hepatocellular carcinoma cells and its regulating effect on the hepatocellular carcinoma invasion and metastasis factors of tissue inhibitor of matrix metalloproteinases-3(TIMP-3)and caveolin-1.Methods:RT-PCR and Western blot were employed to detect the expression of WIF1 in six hepatocellular carcinoma eell lines of HepG2,Hep3 B,Huh7,PLC/PRF/5.SMMC-7721 and MHCC97 and the immortalized human liver cell line THLE-3.Besides,Lipofectamine 2000 was employed to transfect the eukaryotic expression vector pcDNA3.lWIF1 and blank plasmid pcDNA3.1 into hepatocellular carcinoma cell lines.Transwell assay was used to detect the effect of WIF1 on the invasion ability of hepatocellular carcinoma cells;Western blot was used to detect the effect of WIF1 on the expression of TIMP-3 and caveolin-1in hepatocellular carcinoma cells,it also discussed the effect on the expression of β-catenin.Results:The expression of WIF1 in hepatocellular carcinoma cell lines was lower than that in the normal liver cell lines(P<0.01);while there was basically no expression of WIF1 in the human highly metastatic cell line MHCC-97 and moderate expression in HepG2 and SMMC-7721.Therefore,HepG2 and SMMC-7721 were chosen as the further experimental cell lines.After transfecting the eukaryotic expression vector peDNA3.1-WEFl and blank plasmid pcDNA3.1 into hepatocellular carcinoma eell lines,compared with(he blank plasmid group,the cell viability and invasion ability in the WIF1 group were all reduced(P<0.01),the expression of TIMP-3,caveolin-1 and mRNA were all down-regulated(P<0.01),and the expression of β-catenin was decreased(P<0.01).Conclusions:Because of down-regulation or missing of expression of WIFI in hepatocellular carcinoma cell lines,the up-regulation of WIFI expression can significantly inhibit the invasion and metastasis of HepG2 and SMMC-7721 of hepatocellular carcinoma cell lines,which are related to the up-regulated expression of TIMP-3 and down-regulated expression of caveolin-1 and may be realized through the Wnt/β-catenin signaling pathway.
基金This study was supported by the National Natural Science Foundation of China(No.81660360)The Natural Science Foundation of Guangxi(No.2019JJA140605)。
文摘Objective:To observe whether CAV3 is involved in the physiological process of IMGU and NIMGU.Down-regulation of caveolin-3(CAV3)protein expression by small interference RNAs(iRNA)has been studied.Insulin-mediated glucose uptake(IMGU)is critical in skeletal muscle and cardiac myocytes,but non-insulin-mediated glucose uptake(NIMGU)should not be neglected.Methods:CAV3 siRNAs were designed and transfected in C2C12 cells and H9c2 cells in skeletal muscle and cardiac muscle,respectively,and C2C12 and H9c2 cells were cultured in DMEM medium with and without insulin,respectively.Glucose transporter 4(GLUT4)protein expression was detected by Western blot,and the glucose uptake rate of cells was measured by biochemical kit.Results:Transfection with CAV3 siRNA successfully down-regulated CAV3 protein expression in C2C12 and H9c2 cells.In the absence of insulin stimulation,GLUT4 expression was decreased(P<0.01)and glucose uptake was reduced(P<0.05)after 48 h of transfection in C2C12 cells,and GLUT4 expression was decreased(P<0.05)and glucose uptake was reduced(P<0.01)after 48 h of transfection in H9c2 cells.In the presence of insulin stimulation,GLUT4 expression was decreased(P<0.01)and glucose uptake was reduced(P<0.01)after 48 h of transfection in C2C12 cells,and the downregulation of GLUT4 was not statistically significant and glucose uptake was reduced(P<0.01)after 48 hours of transfection in H9c2 cells.Conclusion:Two different states,IMGU and NIMGU,exist in C2C12 cells and H9c2 cells.Both in the quiet state stimulated by insulin as well as in the absence of insulin stimulation,the cellular uptake of glucose is affected by GLUT4 changes regulated by CAV3.
