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
Metabolic syndrome is a pre-diabetic state characterized by several biochemical and physiological alterations,including insulin resistance,visceral fat accumulation,and dyslipidemias,which increase the risk for develo...Metabolic syndrome is a pre-diabetic state characterized by several biochemical and physiological alterations,including insulin resistance,visceral fat accumulation,and dyslipidemias,which increase the risk for developing cardiovascular disease.Metabolic syndrome is associated with augmented sympathetic tone,which could account for the etiology of pre-diabetic cardiomyopathy.This review summarizes the current knowledge of the pathophysiological consequences of enhanced and sustainedβ-adrenergic response in pre-diabetes,focusing on cardiac dysfunction reported in diet-induced experimental models of pre-diabetic cardiomyopathy.The research reviewed indicates that both protein kinase A and Ca^(2+)/calmodulin-dependent protein kinase Ⅱ play important roles in functional responses mediated byβ1-adrenoceptors;therefore,alterations in the expression or function of these kinases can be deleterious.This review also outlines recent information on the role of protein kinase A and Ca^(2+)/calmodulin-dependent protein kinase Ⅱ in abnormal Ca^(2+)handling by cardiomyocytes from diet-induced models of pre-diabetic cardiomyopathy.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
AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immun...AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immunochemistry. Transcriptional and posttranscriptional levels of Ca MKⅡin tissue samples and MMP2,MMP9 and TIMP-1 expression in the human colon cancer cell line HCT116 were assessed by q RTPCR and western blot. Cell proliferation was detected with the MTT assay. Cancer cell migration and invasion were investigated with the Transwell culture system and woundhealing assay.RESULTS We first demonstrated that CaMK Ⅱ was ove rexpressed in human colon cancers and was associated with cancer differentiation. In the human colon cancer cell line HCT116,the Ca MKII-specific inhibitor KN93,but not its inactive analogue KN92,decreased cancer cell proliferation. Furthermore,KN93 also significantly prohibited HCT116 cell migration and invasion. The specific inhibition of ERK1/2 or p38 decreased the proliferation and migration of colon cancer cells.CONCLUSION Our findings highlight Ca MKⅡ as a potential critical mediator in human colon tumor development and metastasis.展开更多
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
The complex of calcineurin B-like protein(CBL)and CBL-interacting protein kinase(CIPK)serves as key components in calcium-sensing,orchestrating various signals crucial for plant growth,development,and responses to bio...The complex of calcineurin B-like protein(CBL)and CBL-interacting protein kinase(CIPK)serves as key components in calcium-sensing,orchestrating various signals crucial for plant growth,development,and responses to biotic and abiotic stresses.However,the mechanism underlying the response of this module to cold stress and its role in flower development in wintersweet(Chimonanthus praecox)remains unclear.Through expression pattern analysis,calcium ion(Ca^(2+))concentration assays,correlation analysis,and linear regression analysis,we found that the[Ca^(2+)],along with CpCBL8 and CpCIPK9 expression levels in wintersweet flower buds(FBs),significantly decreased during the initial flowering stage when the chilling requirement reached 570 chill units(CU).Notably,there was a significant positive correlation between[Ca^(2+)]and CpCBL8 expression.Ca^(2+)increased the expression of Cp CBL8 and CpCIPK9 in FBs,causing a significant delay in the flowering of wintersweet.Furthermore,the function of CpCBL8 was studied using heterologous transformation.Overexpression of CpCBL8 significantly delayed flowering time and significantly reduced cold tolerance and altered the expression pattern of endogenous genes related to low-temperature stress and flower development in transgenic Arabidopsis thaliana.Additionally,transcriptome analysis of chilling-induced dormancy breaking and flower bud enlargement revealed that CpCBL8 and CpCIPK9 were negatively regulated by cold,and the expression pattern of endogenous genes related to flower development and cold stress in wintersweet were similar to that of in A.thaliana.Moreover,protein-protein interaction(PPI)analysis revealed that CpCBL8 and CpCIPK9 interacted in the plasma membrane and nucleus.On the basis of these findings,we speculated that the CpCBL8-CpCIPK9 module plays a crucial role in regulating responses to cold stress and flower development in wintersweet.This study elucidated molecular mechanisms through which the downregulation of the Ca^(2+)-induced CpCBL8-CpCIPK9 module results in dormancy breaking and enhances cold tolerance.This study provides valuable insights for the cultivation of new varieties of wintersweet with increased ornamental value and enhanced cold stress tolerance.展开更多
To explore the mechanism of sperm dysfunction caused by dibutyl phthalate(DBP),the effects of DBP on intracellular[Ca^(2+)]and[pH],reactive oxygen species(ROS),lipid peroxidation(LPO),mitochondrial permeability transi...To explore the mechanism of sperm dysfunction caused by dibutyl phthalate(DBP),the effects of DBP on intracellular[Ca^(2+)]and[pH],reactive oxygen species(ROS),lipid peroxidation(LPO),mitochondrial permeability transition pore(mPTP)opening,mitochondrial membrane potential(MMP),adenosine triphosphate(ATP)levels,phosphorylation of protein kinase A(PKA)substrate proteins and phosphotyrosine(p-Tyr)proteins,sperm motility,spontaneous acrosome reaction,and tail bending were examined in mouse spermatozoa.At 100μg/mL,DBP significantly increased tail bending and[Ca^(2+)]i.Interestingly,DBP showed biphasic effects on[pH]i.DBP at 10–100μg/mL significantly decreased sperm motility.Similarly,Ca^(2+)ionophore A23187 decreased[pH]_(i)sperm motility,suggesting that DBP-induced excessive[Ca^(2+)]_(i)decreased sperm motility.DBP significantly increased ROS and LPO.DBP at 100μg/mL significantly decreased mPTP closing,MMP,and ATP levels in spermatozoa,as did H2O2,indicative of ROS-mediatedmitochondrial dysfunction caused by DBP.DBP as well as H2O2 increased p-Tyr sperm proteins and phosphorylated PKA substrate sperm proteins.DBP at 1–10μg/mL significantly increased the spontaneous acrosome reaction,suggesting that DBP can activate sperm capacitation.Altogether,DBP showed a biphasic effect on intracellular signaling in spermatozoa.At concentrations relevant to seminal ortho-phthalate levels,DBP activates[pH]i,protein tyrosine kinases and PKA via physiological levels of ROS generation,potentiating sperm capacitation.DBP at high doses excessively raises[Ca^(2+)]_(i)and ROS and disrupts[pH]i,impairing the mitochondrial function,tail structural integrity,and sperm motility.展开更多
蛋白质的磷酸化与去磷酸化是细胞信号转导过程中最重要的调控方式,其循环过程就像调控分子的开关一样,参与众多生理活动。负责这一修饰调节的是蛋白激酶与蛋白磷酸酶。报道显示人类染色体编码多达500个蛋白激酶,这些蛋白激酶满足人类高...蛋白质的磷酸化与去磷酸化是细胞信号转导过程中最重要的调控方式,其循环过程就像调控分子的开关一样,参与众多生理活动。负责这一修饰调节的是蛋白激酶与蛋白磷酸酶。报道显示人类染色体编码多达500个蛋白激酶,这些蛋白激酶满足人类高度多样性与差异性调控蛋白磷酸化作用,而有趣的是人类编码的蛋白磷酸酶却仅仅约为150个,其中约有40个是丝氨酸/苏氨酸蛋白磷酸酶。越来越多的证据表明蛋白磷酸酶/蛋白激酶调控异常在心肌病中起关键作用。蛋白磷酸酶1(protein phosphatase 1,PP1)是一多功能的丝氨酸/苏氨酸蛋白磷酸酶,研究显示PP1在心肌肥厚和心衰的发生发展过程中起重要作用。而Ca2+/钙调素依赖性蛋白激酶Ⅱ(Ca2+/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)是一种多功能的丝氨酸/苏氨酸蛋白激酶,它作为Ca2+信号转导的关键因子,调节细胞的多种生物学功能,其功能异常可引起肥厚心肌胞内钙稳态失衡进而引起心律失常等心肌病。该文就PP1与CaMKⅡ的功能和心肌病的关系作一综述。展开更多
目的探讨大鼠骨髓间充质干细胞(rat bone m arrow m esenchym al stem cells,MSCs)体外诱导分化为心肌样细胞内游离钙浓度([Ca2+]i)及钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的表达变化。方法取健康SD大鼠骨髓,用5-氮杂胞苷体外诱导培养。取...目的探讨大鼠骨髓间充质干细胞(rat bone m arrow m esenchym al stem cells,MSCs)体外诱导分化为心肌样细胞内游离钙浓度([Ca2+]i)及钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的表达变化。方法取健康SD大鼠骨髓,用5-氮杂胞苷体外诱导培养。取诱导培养2、3、4周的MSCs为Ⅰ、Ⅱ、Ⅲ组,另取急性分离的心肌细胞为对照组,分别用激光共聚焦技术和W estern b lot技术检测[Ca2+]i及CaMKⅡ表达水平。结果经荧光探针结合Ca2+后,用激光共聚焦技术检测发现,随诱导培养时间的延长,[Ca2+]i逐渐增加;诱导培养4周的MSCs内[Ca2+]i与对照组比较无显著差异[(100.81±17.64),(100.32±17.10),P>0.05]。各组细胞CaMKⅡ的变化趋势与[Ca2+]i定量分析结果相似,Ⅰ、Ⅱ、Ⅲ组及对照组分别为(322.45±19.43)、(434.43±16.77)、(680.91±20.61)、(682.69±21.03),Ⅲ组与对照组比较P>0.05。结论大鼠MSCs在体外诱导培养4周后已分化为心肌样细胞,其细胞内游离钙浓度和CaMKⅡ蛋白表达水平与正常心肌细胞相似。展开更多
Many of the effects of Ca^2＋ signaling are mediated through the Ca^2＋/calmodulin complex and its acceptors, the Ca^2＋/calmodulin-dependent protein kinases, including PSKHI. Studies of the proteins involved in the c...Many of the effects of Ca^2＋ signaling are mediated through the Ca^2＋/calmodulin complex and its acceptors, the Ca^2＋/calmodulin-dependent protein kinases, including PSKHI. Studies of the proteins involved in the calcium metabolism in oysters will help elucidate the pearl formation mechanism. This paper describes a full-length PSKH1 cDNA isolated from pearl oyster Pinctada fucata. Oyster PSKH1 shares 65% homology with human PSKH1 and 48% similarity with rat CaM kinase I in the amino acid sequence, and contains a calmodulin-binding domain. The results of semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization revealed that oyster PSKH1 mRNA is highly expressed in the outer epithelial cells of the mantle pallial and in the gill epithelial cells. These studies provide important information describing the complex Ca^2＋ signaling mechanism in oyster calcium metabolism.展开更多
Objective:Cardiac hypertrophy is an adaptive reaction of the heart against cardiac overloading,but continuous cardiac hypertrophy can lead to cardiac remodeling and heart failure.Cardiac hypertrophy is mostly consider...Objective:Cardiac hypertrophy is an adaptive reaction of the heart against cardiac overloading,but continuous cardiac hypertrophy can lead to cardiac remodeling and heart failure.Cardiac hypertrophy is mostly considered reversible,and recent studies have indicated that decorin not only prevents cardiac fibrosis associated with hypertension,but also achieves therapeutic effects by blocking fibrosis-related signaling pathways.However,the mechanism of action of decorin remains unknown and unconfirmed.Methods:We determined the degree of myocardial hypertrophy by measuring the ratios of the heart weight/body weight and left ventricular weight/body weight,histological analysis and immunohistochemistry.Western blotting was performed to detect the expression levels of CaMKⅡ,p-CaMKⅡ and MEF-2 in the heart.Results:Our results confirmed that decorin can regulate the CaMKⅡ/MEF-2 signaling pathway,with inhibition thereof being similar to that of decorin in reducing cardiac hypertrophy.Conclusion:Taken together,the results of the present study showed that decorin induced cardiac hypertrophy by regulating the CaMKⅡ/MEF-2 signaling pathway in vivo,revealing a new therapeutic approach for the prevention of cardiac hypertrophy.展开更多
Objective:The aim of this study was to investigate the protective effects of ginsenoside Rb1 and assess whether these protective effects are related to calcium/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ).Methods:A ...Objective:The aim of this study was to investigate the protective effects of ginsenoside Rb1 and assess whether these protective effects are related to calcium/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ).Methods:A myocardial ischemia(IS)rat.model and a myocardial H9 C2 cell hypoxia model were established.MI was induced by occluding the left anterior descending artery for 120 min.Ginsenoside Rb1(10 mg/kg)was administered 30 min before ischemia induction,and the treatment continued for 7 days.Results:In the rat IS injury model,ginsenoside Rb1 reduced myocardial infarct size,mean left ventricular diastolic pressure,incidence of arrhythmia,and levels of serum creatine kinase,lactate dehydrogenase,and malondialdehyde.However,the mean left ventricular systolic pressure,and maximal rising and falling rates of ventricular pressure(±dp/dtmax)increased.In the myocardial H9 C2 cell hypoxia model,ginsenoside Rb1 reduced intracellular calcium concentrations([Ca2+]i)during hypoxia,and markedly reversed the hypoxia-induced decrease in cell survival.Ginsenoside Rb1 was involved in the downregulation of CaMKⅡand the ryanodine receptor,as well as hypoxia-induced H9 C2 cell survival.Conclusion:The findings of the present study suggest that ginsenoside Rb1 attenuates MI injury in rats,partially through the downregulation of CaMKⅡexpression.展开更多
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Metabolic syndrome is a pre-diabetic state characterized by several biochemical and physiological alterations,including insulin resistance,visceral fat accumulation,and dyslipidemias,which increase the risk for developing cardiovascular disease.Metabolic syndrome is associated with augmented sympathetic tone,which could account for the etiology of pre-diabetic cardiomyopathy.This review summarizes the current knowledge of the pathophysiological consequences of enhanced and sustainedβ-adrenergic response in pre-diabetes,focusing on cardiac dysfunction reported in diet-induced experimental models of pre-diabetic cardiomyopathy.The research reviewed indicates that both protein kinase A and Ca^(2+)/calmodulin-dependent protein kinase Ⅱ play important roles in functional responses mediated byβ1-adrenoceptors;therefore,alterations in the expression or function of these kinases can be deleterious.This review also outlines recent information on the role of protein kinase A and Ca^(2+)/calmodulin-dependent protein kinase Ⅱ in abnormal Ca^(2+)handling by cardiomyocytes from diet-induced models of pre-diabetic cardiomyopathy.
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.
基金Supported by the National Natural Science Foundation of China,No.81302131
文摘AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immunochemistry. Transcriptional and posttranscriptional levels of Ca MKⅡin tissue samples and MMP2,MMP9 and TIMP-1 expression in the human colon cancer cell line HCT116 were assessed by q RTPCR and western blot. Cell proliferation was detected with the MTT assay. Cancer cell migration and invasion were investigated with the Transwell culture system and woundhealing assay.RESULTS We first demonstrated that CaMK Ⅱ was ove rexpressed in human colon cancers and was associated with cancer differentiation. In the human colon cancer cell line HCT116,the Ca MKII-specific inhibitor KN93,but not its inactive analogue KN92,decreased cancer cell proliferation. Furthermore,KN93 also significantly prohibited HCT116 cell migration and invasion. The specific inhibition of ERK1/2 or p38 decreased the proliferation and migration of colon cancer cells.CONCLUSION Our findings highlight Ca MKⅡ as a potential critical mediator in human colon tumor development and metastasis.
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funded by the Natural Science Foundation of Chongqing(CSTB2023NSCQ-MSX0236)Fundamental Research Funds for the Central Universities(SWU-XDJH202308)Earmarked Funds for the China Agriculture Research System(CARS-26)。
文摘The complex of calcineurin B-like protein(CBL)and CBL-interacting protein kinase(CIPK)serves as key components in calcium-sensing,orchestrating various signals crucial for plant growth,development,and responses to biotic and abiotic stresses.However,the mechanism underlying the response of this module to cold stress and its role in flower development in wintersweet(Chimonanthus praecox)remains unclear.Through expression pattern analysis,calcium ion(Ca^(2+))concentration assays,correlation analysis,and linear regression analysis,we found that the[Ca^(2+)],along with CpCBL8 and CpCIPK9 expression levels in wintersweet flower buds(FBs),significantly decreased during the initial flowering stage when the chilling requirement reached 570 chill units(CU).Notably,there was a significant positive correlation between[Ca^(2+)]and CpCBL8 expression.Ca^(2+)increased the expression of Cp CBL8 and CpCIPK9 in FBs,causing a significant delay in the flowering of wintersweet.Furthermore,the function of CpCBL8 was studied using heterologous transformation.Overexpression of CpCBL8 significantly delayed flowering time and significantly reduced cold tolerance and altered the expression pattern of endogenous genes related to low-temperature stress and flower development in transgenic Arabidopsis thaliana.Additionally,transcriptome analysis of chilling-induced dormancy breaking and flower bud enlargement revealed that CpCBL8 and CpCIPK9 were negatively regulated by cold,and the expression pattern of endogenous genes related to flower development and cold stress in wintersweet were similar to that of in A.thaliana.Moreover,protein-protein interaction(PPI)analysis revealed that CpCBL8 and CpCIPK9 interacted in the plasma membrane and nucleus.On the basis of these findings,we speculated that the CpCBL8-CpCIPK9 module plays a crucial role in regulating responses to cold stress and flower development in wintersweet.This study elucidated molecular mechanisms through which the downregulation of the Ca^(2+)-induced CpCBL8-CpCIPK9 module results in dormancy breaking and enhances cold tolerance.This study provides valuable insights for the cultivation of new varieties of wintersweet with increased ornamental value and enhanced cold stress tolerance.
基金supported by the National Research Foundation of Republic of Korea(NRF)grant funded by the Republic of Korea government(MSIT)(No.2022R1A2C1007831).
文摘To explore the mechanism of sperm dysfunction caused by dibutyl phthalate(DBP),the effects of DBP on intracellular[Ca^(2+)]and[pH],reactive oxygen species(ROS),lipid peroxidation(LPO),mitochondrial permeability transition pore(mPTP)opening,mitochondrial membrane potential(MMP),adenosine triphosphate(ATP)levels,phosphorylation of protein kinase A(PKA)substrate proteins and phosphotyrosine(p-Tyr)proteins,sperm motility,spontaneous acrosome reaction,and tail bending were examined in mouse spermatozoa.At 100μg/mL,DBP significantly increased tail bending and[Ca^(2+)]i.Interestingly,DBP showed biphasic effects on[pH]i.DBP at 10–100μg/mL significantly decreased sperm motility.Similarly,Ca^(2+)ionophore A23187 decreased[pH]_(i)sperm motility,suggesting that DBP-induced excessive[Ca^(2+)]_(i)decreased sperm motility.DBP significantly increased ROS and LPO.DBP at 100μg/mL significantly decreased mPTP closing,MMP,and ATP levels in spermatozoa,as did H2O2,indicative of ROS-mediatedmitochondrial dysfunction caused by DBP.DBP as well as H2O2 increased p-Tyr sperm proteins and phosphorylated PKA substrate sperm proteins.DBP at 1–10μg/mL significantly increased the spontaneous acrosome reaction,suggesting that DBP can activate sperm capacitation.Altogether,DBP showed a biphasic effect on intracellular signaling in spermatozoa.At concentrations relevant to seminal ortho-phthalate levels,DBP activates[pH]i,protein tyrosine kinases and PKA via physiological levels of ROS generation,potentiating sperm capacitation.DBP at high doses excessively raises[Ca^(2+)]_(i)and ROS and disrupts[pH]i,impairing the mitochondrial function,tail structural integrity,and sperm motility.
文摘蛋白质的磷酸化与去磷酸化是细胞信号转导过程中最重要的调控方式,其循环过程就像调控分子的开关一样,参与众多生理活动。负责这一修饰调节的是蛋白激酶与蛋白磷酸酶。报道显示人类染色体编码多达500个蛋白激酶,这些蛋白激酶满足人类高度多样性与差异性调控蛋白磷酸化作用,而有趣的是人类编码的蛋白磷酸酶却仅仅约为150个,其中约有40个是丝氨酸/苏氨酸蛋白磷酸酶。越来越多的证据表明蛋白磷酸酶/蛋白激酶调控异常在心肌病中起关键作用。蛋白磷酸酶1(protein phosphatase 1,PP1)是一多功能的丝氨酸/苏氨酸蛋白磷酸酶,研究显示PP1在心肌肥厚和心衰的发生发展过程中起重要作用。而Ca2+/钙调素依赖性蛋白激酶Ⅱ(Ca2+/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)是一种多功能的丝氨酸/苏氨酸蛋白激酶,它作为Ca2+信号转导的关键因子,调节细胞的多种生物学功能,其功能异常可引起肥厚心肌胞内钙稳态失衡进而引起心律失常等心肌病。该文就PP1与CaMKⅡ的功能和心肌病的关系作一综述。
文摘目的探讨大鼠骨髓间充质干细胞(rat bone m arrow m esenchym al stem cells,MSCs)体外诱导分化为心肌样细胞内游离钙浓度([Ca2+]i)及钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)的表达变化。方法取健康SD大鼠骨髓,用5-氮杂胞苷体外诱导培养。取诱导培养2、3、4周的MSCs为Ⅰ、Ⅱ、Ⅲ组,另取急性分离的心肌细胞为对照组,分别用激光共聚焦技术和W estern b lot技术检测[Ca2+]i及CaMKⅡ表达水平。结果经荧光探针结合Ca2+后,用激光共聚焦技术检测发现,随诱导培养时间的延长,[Ca2+]i逐渐增加;诱导培养4周的MSCs内[Ca2+]i与对照组比较无显著差异[(100.81±17.64),(100.32±17.10),P>0.05]。各组细胞CaMKⅡ的变化趋势与[Ca2+]i定量分析结果相似,Ⅰ、Ⅱ、Ⅲ组及对照组分别为(322.45±19.43)、(434.43±16.77)、(680.91±20.61)、(682.69±21.03),Ⅲ组与对照组比较P>0.05。结论大鼠MSCs在体外诱导培养4周后已分化为心肌样细胞,其细胞内游离钙浓度和CaMKⅡ蛋白表达水平与正常心肌细胞相似。
基金Supported by the National High-Tech Research and Development (863) Program of China (No. 2003AA603430) and the National Natural Science Foundation of China (No. 30371092)
文摘Many of the effects of Ca^2＋ signaling are mediated through the Ca^2＋/calmodulin complex and its acceptors, the Ca^2＋/calmodulin-dependent protein kinases, including PSKHI. Studies of the proteins involved in the calcium metabolism in oysters will help elucidate the pearl formation mechanism. This paper describes a full-length PSKH1 cDNA isolated from pearl oyster Pinctada fucata. Oyster PSKH1 shares 65% homology with human PSKH1 and 48% similarity with rat CaM kinase I in the amino acid sequence, and contains a calmodulin-binding domain. The results of semi-quantitative reverse transcription-polymerase chain reaction and in situ hybridization revealed that oyster PSKH1 mRNA is highly expressed in the outer epithelial cells of the mantle pallial and in the gill epithelial cells. These studies provide important information describing the complex Ca^2＋ signaling mechanism in oyster calcium metabolism.
文摘Objective:Cardiac hypertrophy is an adaptive reaction of the heart against cardiac overloading,but continuous cardiac hypertrophy can lead to cardiac remodeling and heart failure.Cardiac hypertrophy is mostly considered reversible,and recent studies have indicated that decorin not only prevents cardiac fibrosis associated with hypertension,but also achieves therapeutic effects by blocking fibrosis-related signaling pathways.However,the mechanism of action of decorin remains unknown and unconfirmed.Methods:We determined the degree of myocardial hypertrophy by measuring the ratios of the heart weight/body weight and left ventricular weight/body weight,histological analysis and immunohistochemistry.Western blotting was performed to detect the expression levels of CaMKⅡ,p-CaMKⅡ and MEF-2 in the heart.Results:Our results confirmed that decorin can regulate the CaMKⅡ/MEF-2 signaling pathway,with inhibition thereof being similar to that of decorin in reducing cardiac hypertrophy.Conclusion:Taken together,the results of the present study showed that decorin induced cardiac hypertrophy by regulating the CaMKⅡ/MEF-2 signaling pathway in vivo,revealing a new therapeutic approach for the prevention of cardiac hypertrophy.
基金supported by the National Natural Science Funds(81073134)085 First-Class Discipline Construction Innovation Science and Technology Support Project of Shanghai University of TCM(085ZY1206)E-institutes of Shanghai Municipal Education Commission(No E 03008)。
文摘Objective:The aim of this study was to investigate the protective effects of ginsenoside Rb1 and assess whether these protective effects are related to calcium/calmodulin-dependent protein kinaseⅡ(Ca MKⅡ).Methods:A myocardial ischemia(IS)rat.model and a myocardial H9 C2 cell hypoxia model were established.MI was induced by occluding the left anterior descending artery for 120 min.Ginsenoside Rb1(10 mg/kg)was administered 30 min before ischemia induction,and the treatment continued for 7 days.Results:In the rat IS injury model,ginsenoside Rb1 reduced myocardial infarct size,mean left ventricular diastolic pressure,incidence of arrhythmia,and levels of serum creatine kinase,lactate dehydrogenase,and malondialdehyde.However,the mean left ventricular systolic pressure,and maximal rising and falling rates of ventricular pressure(±dp/dtmax)increased.In the myocardial H9 C2 cell hypoxia model,ginsenoside Rb1 reduced intracellular calcium concentrations([Ca2+]i)during hypoxia,and markedly reversed the hypoxia-induced decrease in cell survival.Ginsenoside Rb1 was involved in the downregulation of CaMKⅡand the ryanodine receptor,as well as hypoxia-induced H9 C2 cell survival.Conclusion:The findings of the present study suggest that ginsenoside Rb1 attenuates MI injury in rats,partially through the downregulation of CaMKⅡexpression.
