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
AIM:To investigate the role of caveolin-3(CAV3)and cholecystokinin A receptor(CCKAR)in cholesterol gallstone disease(CGD).METHODS:To establish a mouse model of CGD,male C57BL/6 mice were fed with a lithogenic diet con...AIM:To investigate the role of caveolin-3(CAV3)and cholecystokinin A receptor(CCKAR)in cholesterol gallstone disease(CGD).METHODS:To establish a mouse model of CGD,male C57BL/6 mice were fed with a lithogenic diet containing 1.0%cholic acid,1.25%cholesterol and 15%fat;a similar control group was given a normal diet.The fresh liver weights and liver-to-body weight ratio were compared between the two groups after one month.Serum lipid profile and bile composition were determined with an autoanalyzer.The Cav3 and Cckar mRNA and CAV3and CCKAR protein levels in the liver and gallbladder were determined via real-time polymerase chain reaction and Western blot,respectively.RESULTS:Establishment of the mouse CGD model was verified by the presence of cholesterol gallstones in mice fed the lithogenic diet.Compared with mice maintained on a normal diet,those fed the lithogenic diet had significantly higher mean liver-to-body weight ratio(0.067±0.007 vs 0.039±0.007,P<0.01),serum total cholesterol(4.22±0.46 mmol/L vs 2.21±0.11 mmol/L,P<0.001),bile total cholesterol(1.33±0.33 mmol/L vs 0.21±0.11 mmol/L,P<0.001),and bile phospholipid concentrations(3.55±1.40 mmol/L vs 1.55±0.63 mmol/L,P=0.04),but lower total bile acid concentrations(726.48±51.83μmol/L vs 839.83±23.74μmol/L,P=0.007).The lithogenic diet was also associated with significantly lower CAV3 in the liver and lower CAV3 and CCKAR in the gallbladder compared with the control mice(all P<0.05).CONCLUSION:CAV3 and CCKAR may be involved in cholesterol gallstone disease.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.
基金Supported by National Natural Science Foundation of China,No.81070366
文摘AIM:To investigate the role of caveolin-3(CAV3)and cholecystokinin A receptor(CCKAR)in cholesterol gallstone disease(CGD).METHODS:To establish a mouse model of CGD,male C57BL/6 mice were fed with a lithogenic diet containing 1.0%cholic acid,1.25%cholesterol and 15%fat;a similar control group was given a normal diet.The fresh liver weights and liver-to-body weight ratio were compared between the two groups after one month.Serum lipid profile and bile composition were determined with an autoanalyzer.The Cav3 and Cckar mRNA and CAV3and CCKAR protein levels in the liver and gallbladder were determined via real-time polymerase chain reaction and Western blot,respectively.RESULTS:Establishment of the mouse CGD model was verified by the presence of cholesterol gallstones in mice fed the lithogenic diet.Compared with mice maintained on a normal diet,those fed the lithogenic diet had significantly higher mean liver-to-body weight ratio(0.067±0.007 vs 0.039±0.007,P<0.01),serum total cholesterol(4.22±0.46 mmol/L vs 2.21±0.11 mmol/L,P<0.001),bile total cholesterol(1.33±0.33 mmol/L vs 0.21±0.11 mmol/L,P<0.001),and bile phospholipid concentrations(3.55±1.40 mmol/L vs 1.55±0.63 mmol/L,P=0.04),but lower total bile acid concentrations(726.48±51.83μmol/L vs 839.83±23.74μmol/L,P=0.007).The lithogenic diet was also associated with significantly lower CAV3 in the liver and lower CAV3 and CCKAR in the gallbladder compared with the control mice(all P<0.05).CONCLUSION:CAV3 and CCKAR may be involved in cholesterol gallstone disease.
