Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related.The aim of this study was to de...Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related.The aim of this study was to determine the effects of the calmodulin-dependent protein kinase(CaMK) Ⅱ inhibitor,KN-93,on L-type calcium current(I Ca,L) and early after-depolarizations(EADs) in hypertrophic cardiomyocytes.A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation(LVH group).The control group(sham group) received a sham operation,in which the abdominal aortic was dissected but not coarcted.Eight weeks later,the degree of left ventricular hypertrophy(LVH) was evaluated using echocardiography.Individual cardiomyocyte was isolated through collagenase digestion.Action potentials(APs) and I Ca,L were recorded using the perforated patch clamp technique.APs were recorded under current clamp conditions and I Ca,L was recorded under voltage clamp conditions.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were observed under the conditions of low potassium(2 mmol/L),low magnesium(0.25 mmol/L) Tyrode’s solution perfusion,and slow frequency(0.25-0.5 Hz) electrical stimulation.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92(KN-92 group) and KN-93(KN-93 group).Eight weeks later,the model was successfully established.Under the conditions of low potassium,low magnesium Tyrode’s solution perfusion,and slow frequency electrical stimulation,the incidence of EADs was 0/12,11/12,10/12,and 5/12 in sham group,LVH group,KN-92 group(0.5 μmol/L),and KN-93 group(0.5 μmol/L),respectively.When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group,the incidence of EADs was 10/12 and 2/12,respectively.At 0 mV,the current density was 6.7±1.0 and 6.3±0.7 PA·PF-1 in LVH group and sham group,respectively(P>0.05,n=12).When the drug concentration was 0.5 μmol/L in KN-92 and KN-93 groups,the peak I Ca,L at 0 mV was decreased by(9.4±2.8)% and(10.5±3.0)% in the hypertrophic cardiomyocytes of the two groups,respectively(P>0.05,n=12).When the drug concentration was increased to 1 μmol/L,the peak I Ca,L values were lowered by(13.4±3.7)% and(40±4.9)%,respectively(P<0.01,n=12).KN-93,a specific inhibitor of CaMKII,can effectively inhibit the occurrence of EADs in hypertrophic cardiomyocytes partially by suppressing I Ca,L,which may be the main action mechanism of KN-93 antagonizing the occurrence of ventricular arrhythmias in hypertrophic myocardium.展开更多
Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal ne...Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal neurons to model mi R-219 overexpression.A protective effect of mi R-219 was observed for glutamate-induced neurotoxicity of rat hippocampal neurons,and an underlying mechanism involving calmodulin-dependent protein kinase II γ（Ca MKIIγ） was demonstrated.mi R-219 and Ca MKIIγ m RNA expression induced by glutamate in hippocampal neurons was determined by quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）.After neurons were transfected with mi R-219 mimic,effects on cell viability and apoptosis were measured by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide（MTT） assay and flow cytometry.In addition,a luciferase reporter gene system was used to confirm Ca MKIIγ as a target gene of mi R-219.Western blot assay and rescue experiments were also utilized to detect Ca MKIIγ expression and further verify that mi R-219 in hippocampal neurons exerted its effect through regulation of Ca MKIIγ.MTT assay and q RT-PCR results revealed obvious decreases in cell viability and mi R-219 expression after glutamate stimulation,while Ca MKIIγ m RNA expression was increased.MTT,flow cytometry,and caspase-3 activity assays showed that mi R-219 overexpression could elevate glutamate-induced cell viability,and reduce cell apoptosis and caspase-3 activity.Moreover,luciferase Ca MKIIγ-reporter activity was remarkably decreased by co-transfection with mi R-219 mimic,and the results of a rescue experiment showed that Ca MKIIγ overexpression could reverse the biological effects of mi R-219.Collectively,these findings verify that mi R-219 expression was decreased in glutamate-induced neurons,Ca MKIIγ was a target gene of mi R-219,and mi R-219 alleviated glutamate-induced neuronal excitotoxicity by negatively controlling Ca MKIIγ expression.展开更多
目的:研究cGMP依赖性蛋白激酶Ⅱ(cGMP dependent protein kinaseⅡ,PKGⅡ)对胃癌细胞株BGC-823迁移活动的影响,并对其作用机制进行初步探讨。方法:以携带PKGⅡ基因的腺病毒结构Ad-PKGⅡ感染胃癌BGC-823细胞,用蛋白质印迹法及免疫荧光检...目的:研究cGMP依赖性蛋白激酶Ⅱ(cGMP dependent protein kinaseⅡ,PKGⅡ)对胃癌细胞株BGC-823迁移活动的影响,并对其作用机制进行初步探讨。方法:以携带PKGⅡ基因的腺病毒结构Ad-PKGⅡ感染胃癌BGC-823细胞,用蛋白质印迹法及免疫荧光检测感染病毒后PKGⅡ的表达。用特异性PKGⅡ激活剂8-pCPT-cGMP作用感染病毒的细胞,通过Boyden槽迁移率、刮除迁移分析法分析PKGⅡ高表达和活性增高对Ras同源性蛋白A(Ras homologA,RhoA)激动剂溶血磷脂酸(lysophosphatidic acid,LPA)诱导的胃癌细胞迁移的影响。结果:胃癌细胞株BGC-823在感染Ad-PKGⅡ后高表达PKGⅡ,经cGMP类似物8-pCPT-cGMP激活后,使RhoA活化诱导的细胞迁移活动受到明显抑制,与对照组比较差异有统计学意义(P<0.05)。结论:PKGⅡ能明显抑制胃癌细胞株BGC-823的迁移。展开更多
基金supported by grants from the Fujian Provincial Natural Science Foundation of China (No. 2008J0075)the Fujian Provincial Science and Technology Project of China(No. 2010Y0011)
文摘Cardiac hypertrophy is an independent risk factor for sudden cardiac death in clinical settings and the incidence of sudden cardiac death and ventricular arrhythmias are closely related.The aim of this study was to determine the effects of the calmodulin-dependent protein kinase(CaMK) Ⅱ inhibitor,KN-93,on L-type calcium current(I Ca,L) and early after-depolarizations(EADs) in hypertrophic cardiomyocytes.A rabbit model of myocardial hypertrophy was constructed through abdominal aortic coarctation(LVH group).The control group(sham group) received a sham operation,in which the abdominal aortic was dissected but not coarcted.Eight weeks later,the degree of left ventricular hypertrophy(LVH) was evaluated using echocardiography.Individual cardiomyocyte was isolated through collagenase digestion.Action potentials(APs) and I Ca,L were recorded using the perforated patch clamp technique.APs were recorded under current clamp conditions and I Ca,L was recorded under voltage clamp conditions.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were observed under the conditions of low potassium(2 mmol/L),low magnesium(0.25 mmol/L) Tyrode’s solution perfusion,and slow frequency(0.25-0.5 Hz) electrical stimulation.The incidence of EADs and I ca,L in the hypertrophic cardiomyocytes were also evaluated after treatment with different concentrations of KN-92(KN-92 group) and KN-93(KN-93 group).Eight weeks later,the model was successfully established.Under the conditions of low potassium,low magnesium Tyrode’s solution perfusion,and slow frequency electrical stimulation,the incidence of EADs was 0/12,11/12,10/12,and 5/12 in sham group,LVH group,KN-92 group(0.5 μmol/L),and KN-93 group(0.5 μmol/L),respectively.When the drug concentration was increased to 1 μmol/L in KN-92 group and KN-93 group,the incidence of EADs was 10/12 and 2/12,respectively.At 0 mV,the current density was 6.7±1.0 and 6.3±0.7 PA·PF-1 in LVH group and sham group,respectively(P>0.05,n=12).When the drug concentration was 0.5 μmol/L in KN-92 and KN-93 groups,the peak I Ca,L at 0 mV was decreased by(9.4±2.8)% and(10.5±3.0)% in the hypertrophic cardiomyocytes of the two groups,respectively(P>0.05,n=12).When the drug concentration was increased to 1 μmol/L,the peak I Ca,L values were lowered by(13.4±3.7)% and(40±4.9)%,respectively(P<0.01,n=12).KN-93,a specific inhibitor of CaMKII,can effectively inhibit the occurrence of EADs in hypertrophic cardiomyocytes partially by suppressing I Ca,L,which may be the main action mechanism of KN-93 antagonizing the occurrence of ventricular arrhythmias in hypertrophic myocardium.
基金supported by the National Natural Science Foundation of China,No.81101159the Natural Science Foundation of Jiangsu Province of China,No.BK20151268
文摘Septic encephalopathy is a frequent complication of sepsis,but there are few studies examining the role of micro RNAs（mi Rs） in its pathogenesis.In this study,a mi R-219 mimic was transfected into rat hippocampal neurons to model mi R-219 overexpression.A protective effect of mi R-219 was observed for glutamate-induced neurotoxicity of rat hippocampal neurons,and an underlying mechanism involving calmodulin-dependent protein kinase II γ（Ca MKIIγ） was demonstrated.mi R-219 and Ca MKIIγ m RNA expression induced by glutamate in hippocampal neurons was determined by quantitative real-time reverse transcription-polymerase chain reaction（q RT-PCR）.After neurons were transfected with mi R-219 mimic,effects on cell viability and apoptosis were measured by 3-（4,5-dimethylthiazolyl-2）-2,5-diphenyltetrazolium bromide（MTT） assay and flow cytometry.In addition,a luciferase reporter gene system was used to confirm Ca MKIIγ as a target gene of mi R-219.Western blot assay and rescue experiments were also utilized to detect Ca MKIIγ expression and further verify that mi R-219 in hippocampal neurons exerted its effect through regulation of Ca MKIIγ.MTT assay and q RT-PCR results revealed obvious decreases in cell viability and mi R-219 expression after glutamate stimulation,while Ca MKIIγ m RNA expression was increased.MTT,flow cytometry,and caspase-3 activity assays showed that mi R-219 overexpression could elevate glutamate-induced cell viability,and reduce cell apoptosis and caspase-3 activity.Moreover,luciferase Ca MKIIγ-reporter activity was remarkably decreased by co-transfection with mi R-219 mimic,and the results of a rescue experiment showed that Ca MKIIγ overexpression could reverse the biological effects of mi R-219.Collectively,these findings verify that mi R-219 expression was decreased in glutamate-induced neurons,Ca MKIIγ was a target gene of mi R-219,and mi R-219 alleviated glutamate-induced neuronal excitotoxicity by negatively controlling Ca MKIIγ expression.
