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
OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) us...OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) using wild type and CSE knockout mouse models.METHODS Continuous subcutaneous injection isoprenaline(7.5 mg·kg^(-1) per day),once a day for 4 weeks to induce heart failure in male C57BL/6(6-8 weeks old) mice and CSE-/-mice.150 μmol·L^(-1) H_2O_2 was used to induce oxidative stress in H9c2 cells.Echocardiograph was used to detect cardiac parameters.H&E stain and Masson stain was to observation histopathological changes.Western blot was used to detect protein expression and activity.The si RNA was used to silence protein expression.HPLC was used to detect H_2S level.Biotin assay was used to detect the level of S-sulfhydration protein.RESULTS Treatment with S-propyl-L-cysteine(SPRC) or sodium hydrosulfide(Na HS),modulators of blood H_2S levels,attenuated the development of heart failure in animals,reduced lipid peroxidation,and preserved mitochondrial function.The inhibition Ca MKⅡ phosphorylation by SPRC and Na HS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds.Interestingly,Ca MKⅡ activity was found to be elevated in CSE-/-mice as compared to wild type animals and the phosphorylation status of Ca MK Ⅱ appeared to relate to the severity of heart failure.Importantly,in wild type mice SPRC was found to promote S-sulfhydration of Ca MKⅡ leading to reduced activity of this protein however,in CSE-/-mice S-sulfhydration was abolished following SPRC treatment.CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of Ca MKⅡ is presented.SPRC mediated S-sulfhydration of Ca MKⅡ was found to inhibit Ca MKⅡ activity and to preserve cardiovascular homeostasis.展开更多
钙/钙调蛋白依赖性蛋白激酶Ⅱ(Calcium/Calmodulin-dependent protein kinase Ⅱ,CaMKⅡ)是一种主要表达于心脏中的细胞信号分子,氧化激活的CaMKⅡ与多种致心律失常心脏疾病密切相关。为了分析氧化CaMKⅡ对于细胞电生理的影响,结合CaMK...钙/钙调蛋白依赖性蛋白激酶Ⅱ(Calcium/Calmodulin-dependent protein kinase Ⅱ,CaMKⅡ)是一种主要表达于心脏中的细胞信号分子,氧化激活的CaMKⅡ与多种致心律失常心脏疾病密切相关。为了分析氧化CaMKⅡ对于细胞电生理的影响,结合CaMKⅡ激活的生物化学动力学特征,开发了氧化CaMKⅡ人类心室细胞模型,模拟仿真研究活性氧(Reactive oxygen species,ROS)增强情况下,离子电流、细胞内钙循环和细胞膜动作电位的变化。仿真结果发现,当ROS的浓度升高时,CaMKⅡ的激活量增加,细胞质和肌浆网钙离子浓度升高,动作电位缩短。本模型为研究CaMKⅡ致心律失常提供了量化分析手段,构建了微观分子功能改变与宏观细胞动作电位变化之间的定量关系,具有重要的理论和应用价值。展开更多
目的急性缺血缺氧可致心功能受损和心律失常,钙离子在其中起重要作用,慢性缺氧对心功能及心肌细胞内钙离子活动同样产生影响,但机制不同。该研究拟通过慢性缺氧动物模型,研究慢性缺氧对心肌细胞内钙调素(calmodulin,CaM)、钙/钙调素依...目的急性缺血缺氧可致心功能受损和心律失常,钙离子在其中起重要作用,慢性缺氧对心功能及心肌细胞内钙离子活动同样产生影响,但机制不同。该研究拟通过慢性缺氧动物模型,研究慢性缺氧对心肌细胞内钙调素(calmodulin,CaM)、钙/钙调素依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinase Ⅱ,CaMKⅡ)的表达及其对细胞内Ca2+活动的影响,深入了解慢性缺氧对心脏功能及电活动的影响机制。方法通过吸入低浓度含氧气体(FiO2:10%)建立慢性缺氧大鼠动物模型。在实验1周和3周时,应用RT-PCR,Western Blot方法分别检测对照组和慢性缺氧组动物心肌细胞内CaM和CaMKⅡγ、CaMKIIδmRNA和蛋白表达;分离并培养正常心肌细胞和缺氧3周细胞,应用激光共聚焦法分别检测两种心肌细胞内Ca2+活动,同时应用CaMKⅡ特异性抑制剂KN-62,观察CaMKII在慢性缺氧下对心肌细胞内Ca2+活动的影响。结果实验1周和3周时,慢性缺氧大鼠心肌细胞内CaM和CaMKIIγ、CaMKIIδ的mRNA和蛋白表达均较正常动物高(P<0.01);在缺氧1周和3周组动物间CaM和CaMKIIδ也存在差异(P<0.01),但CaMKⅡγ无差异(P>0.05)。激光共聚焦研究发现,慢性缺氧心肌细胞内Ca2+活动其钙波振幅虽和正常心肌细胞无差异(P>0.05),但钙波时程延长(P<0.01);应用KN-62后,慢性缺氧动物钙波振幅和时程改变较明显(P<0.01)。结论慢性缺氧可使大鼠心肌细胞内CaM和CaMKⅡ合成代偿性增加,保持大鼠心肌细胞内钙稳态,从而在一定时期内维持心功能稳定。但随缺氧时间延长,心功能可受损并可致心律失常。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.
文摘OBJECTIVE To determine the functional role of hydrogen sulfide(H_2S) in protecting against mitochondrial dysfunction in heart failure through the inhibition of Ca^(2+)/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ) using wild type and CSE knockout mouse models.METHODS Continuous subcutaneous injection isoprenaline(7.5 mg·kg^(-1) per day),once a day for 4 weeks to induce heart failure in male C57BL/6(6-8 weeks old) mice and CSE-/-mice.150 μmol·L^(-1) H_2O_2 was used to induce oxidative stress in H9c2 cells.Echocardiograph was used to detect cardiac parameters.H&E stain and Masson stain was to observation histopathological changes.Western blot was used to detect protein expression and activity.The si RNA was used to silence protein expression.HPLC was used to detect H_2S level.Biotin assay was used to detect the level of S-sulfhydration protein.RESULTS Treatment with S-propyl-L-cysteine(SPRC) or sodium hydrosulfide(Na HS),modulators of blood H_2S levels,attenuated the development of heart failure in animals,reduced lipid peroxidation,and preserved mitochondrial function.The inhibition Ca MKⅡ phosphorylation by SPRC and Na HS as demonstrated using both in vivo and in vitro models corresponded with the cardioprotective effects of these compounds.Interestingly,Ca MKⅡ activity was found to be elevated in CSE-/-mice as compared to wild type animals and the phosphorylation status of Ca MK Ⅱ appeared to relate to the severity of heart failure.Importantly,in wild type mice SPRC was found to promote S-sulfhydration of Ca MKⅡ leading to reduced activity of this protein however,in CSE-/-mice S-sulfhydration was abolished following SPRC treatment.CONCLUSION A novel mechanism depicting a role of S-sulfhydration in the regulation of Ca MKⅡ is presented.SPRC mediated S-sulfhydration of Ca MKⅡ was found to inhibit Ca MKⅡ activity and to preserve cardiovascular homeostasis.
文摘目的急性缺血缺氧可致心功能受损和心律失常,钙离子在其中起重要作用,慢性缺氧对心功能及心肌细胞内钙离子活动同样产生影响,但机制不同。该研究拟通过慢性缺氧动物模型,研究慢性缺氧对心肌细胞内钙调素(calmodulin,CaM)、钙/钙调素依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinase Ⅱ,CaMKⅡ)的表达及其对细胞内Ca2+活动的影响,深入了解慢性缺氧对心脏功能及电活动的影响机制。方法通过吸入低浓度含氧气体(FiO2:10%)建立慢性缺氧大鼠动物模型。在实验1周和3周时,应用RT-PCR,Western Blot方法分别检测对照组和慢性缺氧组动物心肌细胞内CaM和CaMKⅡγ、CaMKIIδmRNA和蛋白表达;分离并培养正常心肌细胞和缺氧3周细胞,应用激光共聚焦法分别检测两种心肌细胞内Ca2+活动,同时应用CaMKⅡ特异性抑制剂KN-62,观察CaMKII在慢性缺氧下对心肌细胞内Ca2+活动的影响。结果实验1周和3周时,慢性缺氧大鼠心肌细胞内CaM和CaMKIIγ、CaMKIIδ的mRNA和蛋白表达均较正常动物高(P<0.01);在缺氧1周和3周组动物间CaM和CaMKIIδ也存在差异(P<0.01),但CaMKⅡγ无差异(P>0.05)。激光共聚焦研究发现,慢性缺氧心肌细胞内Ca2+活动其钙波振幅虽和正常心肌细胞无差异(P>0.05),但钙波时程延长(P<0.01);应用KN-62后,慢性缺氧动物钙波振幅和时程改变较明显(P<0.01)。结论慢性缺氧可使大鼠心肌细胞内CaM和CaMKⅡ合成代偿性增加,保持大鼠心肌细胞内钙稳态,从而在一定时期内维持心功能稳定。但随缺氧时间延长,心功能可受损并可致心律失常。
