The effects of BTHP on Ca 2+ independent action potential and the two components of delayed rectifier potassium currents were studied in guinea pig single ventricular myocytes by using whole cell patch clamp tec...The effects of BTHP on Ca 2+ independent action potential and the two components of delayed rectifier potassium currents were studied in guinea pig single ventricular myocytes by using whole cell patch clamp technique. BTHP 30 μmol·L -1 significantly prolonged APD 90 from 143±16 ms to 184±21 ms ( P 【0.01, n=5) without affecting either the RP or APA, and the APD prolonging effects of BTHP were independent of extracellular Ca 2+ . BTHP inhibited both I kr (IC 50 =7 9 μmol·L -1 ) and I ks (IC 50 =22 4 μmol·L -1 ) in a concentration dependent fashion. The results demon strated that BTHP had no obvious selectivity for I kr and I ks .展开更多
Objectives Recent studies have described regional differences in the electrophysiology and pharmacology of ventric- ular myocardium in canine, feline, rat, guinea pig, and human hearts. This has been shown to be due t...Objectives Recent studies have described regional differences in the electrophysiology and pharmacology of ventric- ular myocardium in canine, feline, rat, guinea pig, and human hearts. This has been shown to be due to a smaller IKs and a lager sodium-calcium exchange current (INa-Ca) and late INa in M region ( deep subepicardial to midmyocardial). Studies from our laboratory have found a new repolarization current-nonselective cation current (NSCCs) existing in rabbit fight ventricular myocytes. Methods We examined the characteristics of NSCCs in epicardial, M region, and endocardial cells isolated from the rabbit left ventricle with standard microelectrode and whole-cell patch-clamp tech- niques. The permeability to Na^+ , K^+ , Li^+ , Cs^+ but not to Cl^- indicating that it was a nonselective cation current. Gd^+3 (0. 1 mmol/1) and La^3+ (0. 1 retool/1 ) can block the current markedly. Results Further characterization of NSCCs was significantly smaller in M cells than in epicardial and endocardial cells. NSCCs current density was significantly smaller in M cells than in epicardial and endocardial cells. With repolarization to - 80 mV, INa current density was ( -0. 44 ±0. 05) PA/PF in endocardial cells, ( -0. 12 ±0. 05) PA/PF in M cells and ( - 0. 28 ±0. 07) PA/PF in epicardial cells ; and with repolarization to + 30 mV, INa, current density was ( 1.09 ± 0. 29) PA/PF in endocardial cells, (0. 38±0. 09) PA/PF in M cells and (0. 91 ± 0. 32) PA/PF in epicardial cells. Conclusions Transmural dispersion of repolarization was due to the heterogeneity of NSCCs in rabbit left ventricle epicardial, endocardial myocytes and M cells. These findings may advance our understanding of the ionic basis for our understanding of factors contributing to the development of cardiac arrhythmias.展开更多
Objective:To investigate the protective function of tocilizumab in human cardiac myocytes ischemia-reperfusion injury.Methods:The human cardiac myocytes were treated by tocilizumab with different concentrations(1.0 mg...Objective:To investigate the protective function of tocilizumab in human cardiac myocytes ischemia-reperfusion injury.Methods:The human cardiac myocytes were treated by tocilizumab with different concentrations(1.0 mg/mL,3.0 mg/mL,5.0 mg/mL) for 24 h.then cells were cultured in ischemia environment for 24 h and reperfusion environment for 1 h.The MTT and flow cytometry were used to detect the proliferation and apoptosis of human cardiac myocytes,respectively.The mRNA and protein expressions of Bcl-2 and Bax were measured by qRT-PCR and western blot,respectively.Results:Compared to the negative group,pretreated by tocilizumab could significantly enhance the proliferation viability and suppress apoptosis of human cardiac myocytes after suffering ischemia reperfusion injury(P<0.05).The expression of Bcl-2 in tocilizumab treated group were higher than NC group(P<0.05).while the Bax expression were lower(P<0.05).Conclusions:Tocilizumab could significantly inhibit apoptosis and keep the proliferation viability of human cardiac myocytes after suffering ischemia reperfusion injury.Tocilizumab may obtain a widely application in the protection of ischemia reperfusion injury.展开更多
Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion ...Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode's solution, then with Ca 2+-free Tyrode's solution and subsequently with low Ca 2+ enzyme solution containing collagenase 0.1-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37 ℃. Finally hearts were washed by Ca 2+-free Tyrode's solution, after which the ventricles were minced into small pieces in KB solution, dispersed and filtered. The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp experiments.Results: When all the factors such as water, enzyme, Ca 2+,pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30%-50% came out.Conclusion: All the factors should be well controlled, which ensured the isolated cells Ca 2+ tolerant and appropriate for patch clamp experiments.展开更多
Intermittent hypoxia has been shown to provide myocardial protection against ishemia/reperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to i...Intermittent hypoxia has been shown to provide myocardial protection against ishemia/reperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts compared with normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reduce myocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl-2/Bax, especially in membrane fraction.展开更多
This study examined the effect of cholic acid (CA) on cultured cardiac myoeytes (CMs) from neonatal rats with an attempt to explore the possible mechanism of sudden fetal death in intra- hepatic cholestasis of pre...This study examined the effect of cholic acid (CA) on cultured cardiac myoeytes (CMs) from neonatal rats with an attempt to explore the possible mechanism of sudden fetal death in intra- hepatic cholestasis of pregnancy (ICP). Inverted microscopy was performed to detect the impact of CA on the beating rates of rat CMs. MTT method was used to study the effect of CA on the viability of CMs. CMs cultured in vitro were incubated with 10 ~maol/L Ca2+-sensitive fluorescence indicator fluo-3/AM. The fluorescence signals of free calcium induced by CA were measured under a laser scanning confocal microscope. The results showed that CA decreased the beating rates of the CMs in a dose-dependent manner. CA could suppress the activities of CMs in a time- and dose-dependent manner. CA increased the concentration of intracellular free calcium in a dose-dependent manner. Our study suggested that CA could inhibit the activity of CMs by causing calcium overload, thereby leading to the sudden fetal death in ICP.展开更多
Objective To investigate the effect of peroxisome proliferator-activated receptor-α(PPARα) and PPARγactivators on tumor necrosis factor-α(TNFα) expression in neonatal rat cardiac myocytes. Methods Primary culture...Objective To investigate the effect of peroxisome proliferator-activated receptor-α(PPARα) and PPARγactivators on tumor necrosis factor-α(TNFα) expression in neonatal rat cardiac myocytes. Methods Primary cultures of cardiac myocytes from 1- to 3-day-old Wistar rats were prepared, and myocytes were ex-posed to lipopolysaccharide (LPS) and varying concentrations of PPARαor PPARγactivator (fenofibrate or pioglitazone).RT-PCR and ELISA were used to measure TNFα, PPARα, and PPARγexpression in cultured cardiac myocytes. Transient tr-ansfection of TNFαpromoter with or without nuclear factor-kappaB (NF-κB) binding site to cardiac myocytes was performed. Results Pretreatment of cardiac myocytes with fenofibrate or pioglitazone inhibited LPS-induced TNFαmRNA and protein expression in a dose-dependent manner. However, no significant changes were observed on PPARαor PPARγmRNA expression when cardiac myocytes were pretreated with fenofibrate or pioglitazone. Proportional suppression of TNFαpromoter activity was observed when myocytes was transiently transfected with whole length of TNFαpromoter (-721/+17) after being stimulated with LPS and fenofibrate or pioglitazone, whereas no change of promoter activity was observed with transfection of TNFαreporter construct in deletion of NF-κB binding site (-182/+17). Conclusions PPARαand PPARγactivators may inhibit cardiac TNFαexpression but not accompanied by change of PPARαor PPARγmRNA expression. Therefore PPARαand PPARγactivators appear to play a role in anti-inflammation. The mechanism may partly be involved in suppression of the NF-κB pathway.展开更多
AIM: To investigate the effects of exogenous unsaturated fatty acids on calcium-activated potassium current [IK(Ca)] in gastric antral circular myocytes of guinea pigs. METHODS: Gastric myocytes were isolated by colla...AIM: To investigate the effects of exogenous unsaturated fatty acids on calcium-activated potassium current [IK(Ca)] in gastric antral circular myocytes of guinea pigs. METHODS: Gastric myocytes were isolated by collagenase from the antral circular layer of guinea pig stomach. The whole-cell patch clamp technique was used to record /K(Ca) in the isolated single smooth muscle cells with or without different concentrations of arachidonic acid (AA), linoleic acid (LA), and oleic acid (OA). RESULTS: AA at concentrations of 2,5 and 10 μmol/L markedly increased IK(Ca) in a dose-dependent manner. LA at concentrations of 5, 10 and 20 μmol/L also enhanced /K(Ca) in a dose-dependent manner. The increasing potency of AA, LA, and oleic acid (OA) on /K(Ca)at the same concentration (10μmol/L) was in the order of AA>LA>OA. AA (10 μmol/L)-induced increase of Ik(ca) was not blocked by H-7 (10 μmol/L), an inhibitor of protein kinase C (PKC), or indomethacin (10 μmol/L), an inhibitor of the cyclooxygenase pathway, and 17-octadecynoic acid (10 μmol/L), an inhibitor of the cytochrome P450 pathway, but weakened by nordihydroguaiaretic acid (10μmol/L), an inhibitor of the lipoxygenase pathway. CONCLUSION: Unsaturated fatty acids markedly increase Ik(Ca), and the enhancing potencies are related to the number of double bonds in the fatty acid chain. The lipoxygenase pathway of unsaturated fatty acid metabolism is involved in the unsaturated fatty acid-induced increase of IK(Ca) in gastric antral circular myocytes of guinea pigs.展开更多
The whole-cell patch-clamp technique was employed to obtain information about the voltage-dependence and kinetics of interaction of 7-chlor-benzylte-trahydropalmatine (7-Cl-BTHP) with cardiac sodium channels. 7-Cl-BTH...The whole-cell patch-clamp technique was employed to obtain information about the voltage-dependence and kinetics of interaction of 7-chlor-benzylte-trahydropalmatine (7-Cl-BTHP) with cardiac sodium channels. 7-Cl-BTHP (30 mol/L) significantly decreased the peak sodium current (from 7. 8±1. 8 nA to 5. 3±1. 4 nA, P<0. 01, n=5), without producing a shift of the current-voltage curve. It shifted the inactivation curves of sodium current to hyperpolarized potentials, and the V(0.5) was shifted from - (82. 5±2. 5) mV to - (95±2.4) mV (P <0. 05, n=4). 7-Cl-BTHP produced a significant use-dependent effect that was proportional to the duration of the voltage step. In addition, 7-Cl-BTHP slowed the recovery of sodium channel from inactivation, which could explain its use-dependent effects on sodium current. The characteristics of 7-Cl-BTHP blockage suggest that this agent binds preferentially to inactivated sodium channels.展开更多
Objective To investigate the effects of adenosine (Ado) on myocardiac electrophysiology in simu- lated ischemla and reperfusion in guinea-pig ventricular myocytes. Methods Electrical activity was recorded using stan...Objective To investigate the effects of adenosine (Ado) on myocardiac electrophysiology in simu- lated ischemla and reperfusion in guinea-pig ventricular myocytes. Methods Electrical activity was recorded using standard intracellular microelectrode technique. Right ventricle was superfused with simulated ischemic Tyrode’s so- lution for 15 min, and reperfued with normal Tyrode’s solution for 30 min. Results The results showed Ado had no measurable effects on guinea-pig ventricular myocytes in normal Tyrode’s solution. In the presence of Ado, maximal diastolic potential tended to be more depolarized during ischemia, and action potential (AP) parameters were abbrevi- ated greatly in a concentration-dependent manner. Especially, the concentration of Ado 100 μmol·L-1 had significant effects on AP parameters in ischemic phase [APD30, APD50, and APD90 reduced by (86±8)% versus (65±6)%, (70 ±7)% versus (50±6)%, and (60±6)% versus (42±4)% for control after 15 min, P<0.O5]. During reperfu- sion, AP parameters did not completely return to initial values in presence of Ado. This study illustrated that Ado significantly decreased incidence of arrhythmias induced by ischemia and reperfusion (in presence of Ado 100 μmol· L-1, the incidence of DAD decreased by 17% versus 82% for control during reperfusion). Conclusion Ado has no significant effects on guinea-pig ventricle in normal conditions, abbreviates greatly AP parameters during ischemia with a concentration-dependent manner, and has marked antiarrhythmic effects in ischemia and reperfusion.展开更多
Objectives To investigate the anti-apoptotic effects of mesenchymal stem cells (MSCs) on hypoxic injured cardiac myocytes in vitro. Methods MSCs were isolated from bone marrow of Sprague-Dawley (SD) rats, and card...Objectives To investigate the anti-apoptotic effects of mesenchymal stem cells (MSCs) on hypoxic injured cardiac myocytes in vitro. Methods MSCs were isolated from bone marrow of Sprague-Dawley (SD) rats, and cardiac myocytes from neonatal rats. The rat cardiac myocytes were co-cultured with MSCs or MSC-conditioned media in anoxia (95% N2 ±5% CO2) for 72 hours. Cell apoptosis was measured by Hoechst 33258 staining. The expression of Bcl-2 and Bax in cardiac myocytes was tested by Western Blot. Results The apoptotic rate was 51.6% ± 2.4% when cardiac myocytes were cultured in continuous hypoxia and was significantly decreased when cardiac myocytes were cocultured with MSCs or MSC-conditioned media ( 15.1% ± 5.4% and 24. 0% ± 4.2% respectively, P 〈 0. 001 ). The decreased expression of Bax in the cardiac myocytes was greatly related to the decreasing of apoptosis, but there was no difference in Bcl-2 expression among these groups. Conclusions Co-cultured MSCs showed significant anti-apoptotic effects on cardiac myocytes in continuous hypoxia. The mechanism may be the interact of cell to cell and paracrine of cytokines which effected the expression of Bax in the cardiac myocytes.展开更多
Objective To explore the effect of allocryptopine (All) on the Late sodium current (INa,Late) of atrial myocytes in spontaneously hyper- tensive rats (SHR). Method The enzyme digestion method was used to separat...Objective To explore the effect of allocryptopine (All) on the Late sodium current (INa,Late) of atrial myocytes in spontaneously hyper- tensive rats (SHR). Method The enzyme digestion method was used to separate single atrial myocytes from SHR and Wistar-Kyoto rat (WKY) rats. INa,Late was record by patch-clamp technique and the effect of All on the current was evaluated. Results Comparing with WKY cells, markedly increasing of INa,Late current in SHR myocytes was found from 0.24 ± 0.02 pA/pF of WKY cells to 1.73± 0.04 pA/pF of SHR cells (P 〈 0.01, n = 15). After treament with 30 μmol/L All; the current densities was reduced to 0.92 ± 0.03 pA/pF. The ratio of INa,Late/INa,peak of WKY and SHR were 0.09% ± 0.01% and 0.71% ± 0.02%, INa, Late/INa,peak of SHR was reduced to 0.37% ± 0.02% by 30 μmol/L All (P 〈 0.01, n = 15). We also determined the effect of All on the gating mechanism of the INa,Late in the SHR cells. It was found that All decreased the INa,Late by alleviating the inactivation of the channels and increasing the window current of sodium channel. Conclusion Increased INa,Late in SHR atrial myocytes and the prolonged APD were inhibited by All coming from Chinese herb medicine.展开更多
Objectives To analyze and identify differentially expressed phosphoproteins associated with mitochondrial KATP channel opening. Methods: Adult rat ventricular myocytes were isolated, cultured, and identified, and pre...Objectives To analyze and identify differentially expressed phosphoproteins associated with mitochondrial KATP channel opening. Methods: Adult rat ventricular myocytes were isolated, cultured, and identified, and pretreated without or with 100 μmol/L diazoxide for 10 min. Phosphoproteins prepared and enriched from the control and diazoxide-pretreated cells were separated by two-dimensional gel electrophoresis (2-DE) followed by sliver staining. The obtained interesting phosphoproteins were further identified by mass spectrometry. Results. Associated with diazoxide preconditioning, the proteins of chaperonin containing TCP-1 and hypothetical protein XP-346548 were phosphorylated significantly (P〈0. 01), while the 94-kDa glucose-regulated protein, calpactin I heavy chain and ferritin were dephosphorylated markedly (P〈0. 01). Conclusion: These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins in order to respond diazoxide preconditioning, and these phosphorylated protein may mediate the downstream signaling of cardioprotection by mitochondrial KATp channel opening induced by ischemic preconditioning.展开更多
Objective To observe the anti-CVB3 ( Coxsackievirus B3 ) effect of sophocarpine (SC) extracted from Sophora flavescens, a traditional Chinese herb in vitro. Methods Cardiomyocytes from the neonatal rat were cultur...Objective To observe the anti-CVB3 ( Coxsackievirus B3 ) effect of sophocarpine (SC) extracted from Sophora flavescens, a traditional Chinese herb in vitro. Methods Cardiomyocytes from the neonatal rat were cultured to establish the viral myocarditis model The cells were divided into four groups: infected group ( infected by CVB3 ) , SC treated group ( added SC 100 μg/mL after viral infection ), SC control group ( added SC 100 μg/mL only), and normal control group. The cytopathic effect (CPE) and the beating frequency of the myocardial cells were observed and the LDH levels in the supernatant were measured at day 2,3, and 5. The cultured myocytes were added different concentrations of SC ( 12. 5 -400 μg/mL ) after infection with CVB3, the CPE was observed and the concentrations of LDH were measured and compared at day 2, 3, and 5. Results In the SC treated group ( 100 μg/mL ) , the cytopathic effect was lighter and the LDH level was lower than the infected group. SC in a concentration of 12. 5 - 300 μg/mL could relieve the CPE and lower the LDH level, while in a higher concentration (400 μ/m ) , it exacerbated the CPE caused by the virus, and the LDH levels were higher than the infected cells. Conclusion SC in certain concentration could protect the cultured rat cardiomyocytes from CVB3 infection.展开更多
Reactive oxygen species(ROS) plays a key role in human heart diseases. Glutathione peroxidase(GPX) functions as an antioxidant as it catalyzes the reduction of hydroperoxide. In order to investigate the antioxidan...Reactive oxygen species(ROS) plays a key role in human heart diseases. Glutathione peroxidase(GPX) functions as an antioxidant as it catalyzes the reduction of hydroperoxide. In order to investigate the antioxidant effect of human selenium-containing single-chain Fv(Se-scFv-B3), a new mimic of GPX, a model system of hydrogen peroxide(H202)-induced rat cardiac myocyte damage was established. The cardiac myocyte damage was characte- rized in terms of cell viability, lipid peroxidation, cell membrane integrity, and intracellular H202 level. The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability, the decline of malondialdehyde(MDA) production, lactate dehydrogenase(LDH) release, and intracellular H2O2 level. So Se-scFv-B3 may have a great potential in the treatment of human heart diseases induced by ROS.展开更多
Bone marrow mesenchymal stem cells (BMSCs) could differentiate into various cell types including adipocytes and myocytes, which had important scientific significance not only in the field of tissue regeneration, but...Bone marrow mesenchymal stem cells (BMSCs) could differentiate into various cell types including adipocytes and myocytes, which had important scientific significance not only in the field of tissue regeneration, but also in the field of agricultural science. In an attempt to exhibit the characterization and differentiation into adipocytes and myocytes of porcine BMSCs, we isolated and purified porcine BMSCs by red blood cell lysis method and percoll gradient centrifugation. The purified cells presented a stretched fibroblast-like phenotype when adhered to the culture plate. The results of flow cytometry analysis and immunofluorescence staining demonstrated that the isolated cells were positive for mesenchymal surface markers CD29, CD44 and negative for hematopoietic markers CD45 and the adhesion molecules CD31. Cells were induced to differentiate into adipocytes with adipogenic medium containing insulin, dexamethasone, oleate and octanoate. Oil Red O staining demonstrated that the porcine BMSCs successfully differentiated to adipocytes. Moreover, the findings of real-time PCR and Western blotting indicated that the induced cells expressed adipogenic marker genes (PPAR-y, C/EBP-c~, perilipin, aP2) mRNA or proteins (PPAR-3,, perilipin, aP2). On the other hand, porcine BMSCs were induced into myoctyes with myogenic medium supplemented with 5-azacytidine, basic fibroblast growth factor, chick embryo extract and horse serum. Morphological observation by hochest 33342 staining showed that the induced cells presented as multi-nucleus muscular tube structure. And myogenic marker genes (Myf5, desmin) mRNA or proteins (MyfS, MyoD, myogenin, desmin) were found in the induced cells. In addition, the results of immunofluorescence staining revealed that myogenic marker (Myf5, MyoD, myogenin, desmin, S-MyHC) proteins was positive in the induced cells. Above all, these results suggested that the isolated porcine BMSCs were not only consistent with the characterization of mesenchymal stem cells, but also exhibited the multipotential capacity to form adipocytes and myocytes, which provided the basis to investigate the regulation mechanism involved in the selective differentiation of porcine BMSCs.展开更多
As one of the most important antioxidant enzymes, glutathione peroxidase(GPX) protects cells and tissues from oxidative damage, and plays an important role in cardiovascular and cerebrovascular injuries induced by o...As one of the most important antioxidant enzymes, glutathione peroxidase(GPX) protects cells and tissues from oxidative damage, and plays an important role in cardiovascular and cerebrovascular injuries induced by oxida- tive stress. The antioxidant effect of selenium-containing glutathione S-transferase(Se-GST), a mimic of GPX was investigated on rat cardiomyocytes. To explore the protection function of Se-GST in hydrogen peroxide(H202) chal- lenged rat cardiomyocytes, we examined malondialdehyde(MDA), lactate dehydrogenase(LDH), superoxide dismu- tase(SOD) and cell apoptosis. The results demonstrate exposure of rat cardiomyocytes to H202 for 6 and 12 h induced the significant increases of MDA, LDH and apoptosis rate of cardiomyocytes, but pretreatment of rat cardiomyocytes with Se-GST at 0.0005 or 0.001 unit/mL prevents oxidative stress induced by H202 with the decreases of cell apopto- sis. All the results hint Se-GST has antioxidant activity for oxidative stress challenged rat cardiomyocvtes.展开更多
Objective:To observe the protective effects of erythropoietin (EPO) pretreatment on cardiac myocyte with hypoxia/reoxygenation (H/R) injury and the role of NF-κBin this effects. Methods:After the H/R model of c...Objective:To observe the protective effects of erythropoietin (EPO) pretreatment on cardiac myocyte with hypoxia/reoxygenation (H/R) injury and the role of NF-κBin this effects. Methods:After the H/R model of cardiac myocytes of neonatal rats was established, the cultured cardiac myocytes were divided into 4 groups, including EPO pretreatment group ( EPO 10 U/ml 24 h before H/R), EPO pretreatment + PDTC group(EPO 10 U/ml and PDTC 5 μg/ml 24 h before H/R), PDTC group (PDTC 5 μg /ml 24 h before H/R) and eomrolgroup. Before and after the H/R, assay of LDH concentration in the culture medium, the survival rate of the myocytes tested by MTT chromatometry and the apoptosis by flow cytometry were undertaken. Activation of NF-κB was determined by EMSA before and after H/R. Results:EPO pretreatment markedly reduced the LDH concentration in the medium, elevated the survival rate of myocytes and inhibited the apoptosis after H/R. Addition of PDTC during the pretreatment abol- ished the protective effects of EPO pretreatment. NF-κB was markedly activated during EPO pretreatment and PDTCinhibited the activation. However, after H/R, the activity of NF-κB in myocytes with EPO pretreatment was significantly inhibited compared to the other myocytes. Conclusion:NF-κB is significantly activated during EPO pretreatment, but is inhibited after H/R, which is correlated with the protective effects of EPO pretreatment on cardiac myocytes with H/R. This phenomenon can be explained as the negative feedback mechanism of the activation of NF-κB.展开更多
Objectives To investigate the effects of β2-adrenergic antagonist on cytosolic Ca^2 + ([Ca^2+ ]i) in ventricular myocytes from infarcted rat heart. Methods A ligature was placed around left anterior descending co...Objectives To investigate the effects of β2-adrenergic antagonist on cytosolic Ca^2 + ([Ca^2+ ]i) in ventricular myocytes from infarcted rat heart. Methods A ligature was placed around left anterior descending coronary artery of rat hearts. Rats in the control group were sham-operated. Cardiomyocytes were dissociated at two, four, eight weeks after myocardial infarction (MI) and [Ca^2+]i was measured via fura-2 fluorescence. The response of cardiomyocytes to isoproterenol in presence or absence of betal-adrenergic antagonist atenolol, beta2-adrenergic antagonist ICI118, 551 or non-selective β1, 2- adrenergic antagonists propranolol was examined. Results The followings were found that ICI 118, 551 had no significant effects on the rise of [Ca^2+]i induced by isoproterenol in normal ventricular myocytes (P 〉 0.05), ICI118, 551 only significantly attenuated the rise of [Ca^2+]i induced by isoproterenol at four weeks and eight weeks after MI (24.5%±5.7% vs 57.8% ± 13.2%, P〈 0.01; 12.2%±7.9% vs 44.6%±11.3%, P〈 0.01). Atenolol had suppressive effects only in the control group and the post-MI group of two weeks (P 〈 0.05), and propranolol had suppressive effects in the control and all the three post-MI groups (P 〈 0.01). Conclusions Beta2-adrenergic antagonist ICI118, 551 may exert negative effects on Ca^2+ overload initiated by sympathetic stimulation after MI.展开更多
Objective To study the effects of simvastatin on the hypertrophy of cultured rat cardiac myocytes induced by serum and the role ofphosphatase and tensin homolog deleted on chromosome ten (PTEN) in the signal pathway...Objective To study the effects of simvastatin on the hypertrophy of cultured rat cardiac myocytes induced by serum and the role ofphosphatase and tensin homolog deleted on chromosome ten (PTEN) in the signal pathway. Methods Cultured neonatal Sprague- Dawley (SD) rat cardiac myocytes were treated with 15% fetal bovine serum, or without serum, or different consentrations of simvastatin. Image analysis system was used to measure the cardiac myocytes surface area. Protein synthesis of myocytes was measured via [3H]-leucine incorporation method. The expression level of atrial natriuretic peptide (ANP) mRNA in myocytes was determined with reverse transcription polymerase chain reaction (RT-PCR). The mRNA and protein expression levels of PTEN in cardiac myocytes were investigated with RT-PCR and Western blot respectively. Results At 24 hours, cardiac myocytes surface area was significantly higher in 15% serum group (1611.16± 160.75 lam2) than in serum-free group (538.04±118.60 ±tm2, P〈0.01). Simvastatin decreased the cell surface area in a concentration dependent manner. The cell surface area in 10-5 and 10-6 mol/L simvastatin groups were 799.84+ 167.70 ±tm2 and 1076.88± 199.28 um2 respectively, which were both significantly lower than that in 15% fetal bovine serum group (P〈0.01). Incorporation rate of [3H]-leucine was significantly higher in 15% fetal bovine serum group (2360± 106cpm/well) than that in serum-free group (1305±92 cpm/well, P〈0.01). Incorporation rate of [3H]-leucine in 10.5 and 10.6 mol/L simvastatin groups were 1707±101 clam/well and 1962±125 cpm/well respectively, which were both lower than that in serum group (P〈0.01). With the increase of simvastatin concentration, the expression level ofANP mRNA in cardiac myocytes was decreased gradually, which were 0.29±0.03 and 0.40-±0.03 respectively in 10.5 and 10-6 mol/L simvastatin groups, and significantly lower than that in serum group(0.60-±.03, P〈0.01). Simvastatin increased the expressions of PTEN mRNA and protein in cardiac myocytes in a concentration dependent manner. PTEN mRNA expression level in 10-7, 10-6and 105mol/L simvastatin groups were 0.38±0.03, 0.83±0.04 and 0.85±0.05, respectively, which were all higher than that in 15% fetal bovine serum group (0.29±0.04, P〈0.05). Similarly, PTEN protein level in 10-7, 10-6 and 10.5 mol/L simvastatin groups (39.25±3.41, 46.35±1.78 and 47.22±2.39 respectively) were also significantly higher than that in 15% fetal bovine serum group (32.21±4.06, P〈0.05). Conclusion Simvastatin can inhibit the hypertrophy of cultured rat cardiac myoeytes induced by serum, and the increase of expression level of PTEN might be involved in the mechanism (J Geriatr Cardio12010; 7:47-51).展开更多
文摘The effects of BTHP on Ca 2+ independent action potential and the two components of delayed rectifier potassium currents were studied in guinea pig single ventricular myocytes by using whole cell patch clamp technique. BTHP 30 μmol·L -1 significantly prolonged APD 90 from 143±16 ms to 184±21 ms ( P 【0.01, n=5) without affecting either the RP or APA, and the APD prolonging effects of BTHP were independent of extracellular Ca 2+ . BTHP inhibited both I kr (IC 50 =7 9 μmol·L -1 ) and I ks (IC 50 =22 4 μmol·L -1 ) in a concentration dependent fashion. The results demon strated that BTHP had no obvious selectivity for I kr and I ks .
文摘Objectives Recent studies have described regional differences in the electrophysiology and pharmacology of ventric- ular myocardium in canine, feline, rat, guinea pig, and human hearts. This has been shown to be due to a smaller IKs and a lager sodium-calcium exchange current (INa-Ca) and late INa in M region ( deep subepicardial to midmyocardial). Studies from our laboratory have found a new repolarization current-nonselective cation current (NSCCs) existing in rabbit fight ventricular myocytes. Methods We examined the characteristics of NSCCs in epicardial, M region, and endocardial cells isolated from the rabbit left ventricle with standard microelectrode and whole-cell patch-clamp tech- niques. The permeability to Na^+ , K^+ , Li^+ , Cs^+ but not to Cl^- indicating that it was a nonselective cation current. Gd^+3 (0. 1 mmol/1) and La^3+ (0. 1 retool/1 ) can block the current markedly. Results Further characterization of NSCCs was significantly smaller in M cells than in epicardial and endocardial cells. NSCCs current density was significantly smaller in M cells than in epicardial and endocardial cells. With repolarization to - 80 mV, INa current density was ( -0. 44 ±0. 05) PA/PF in endocardial cells, ( -0. 12 ±0. 05) PA/PF in M cells and ( - 0. 28 ±0. 07) PA/PF in epicardial cells ; and with repolarization to + 30 mV, INa, current density was ( 1.09 ± 0. 29) PA/PF in endocardial cells, (0. 38±0. 09) PA/PF in M cells and (0. 91 ± 0. 32) PA/PF in epicardial cells. Conclusions Transmural dispersion of repolarization was due to the heterogeneity of NSCCs in rabbit left ventricle epicardial, endocardial myocytes and M cells. These findings may advance our understanding of the ionic basis for our understanding of factors contributing to the development of cardiac arrhythmias.
基金supported by a grant from the Health Department Foundation of Zhejiang Province(2010KYA102)
文摘Objective:To investigate the protective function of tocilizumab in human cardiac myocytes ischemia-reperfusion injury.Methods:The human cardiac myocytes were treated by tocilizumab with different concentrations(1.0 mg/mL,3.0 mg/mL,5.0 mg/mL) for 24 h.then cells were cultured in ischemia environment for 24 h and reperfusion environment for 1 h.The MTT and flow cytometry were used to detect the proliferation and apoptosis of human cardiac myocytes,respectively.The mRNA and protein expressions of Bcl-2 and Bax were measured by qRT-PCR and western blot,respectively.Results:Compared to the negative group,pretreated by tocilizumab could significantly enhance the proliferation viability and suppress apoptosis of human cardiac myocytes after suffering ischemia reperfusion injury(P<0.05).The expression of Bcl-2 in tocilizumab treated group were higher than NC group(P<0.05).while the Bax expression were lower(P<0.05).Conclusions:Tocilizumab could significantly inhibit apoptosis and keep the proliferation viability of human cardiac myocytes after suffering ischemia reperfusion injury.Tocilizumab may obtain a widely application in the protection of ischemia reperfusion injury.
文摘Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode's solution, then with Ca 2+-free Tyrode's solution and subsequently with low Ca 2+ enzyme solution containing collagenase 0.1-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37 ℃. Finally hearts were washed by Ca 2+-free Tyrode's solution, after which the ventricles were minced into small pieces in KB solution, dispersed and filtered. The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp experiments.Results: When all the factors such as water, enzyme, Ca 2+,pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30%-50% came out.Conclusion: All the factors should be well controlled, which ensured the isolated cells Ca 2+ tolerant and appropriate for patch clamp experiments.
基金The study was supported by grants from National Natural Science Foundation of Chinathe Science and Technology committee of Shanghai Municipality(02JC14038).
文摘Intermittent hypoxia has been shown to provide myocardial protection against ishemia/reperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts compared with normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reduce myocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl-2/Bax, especially in membrane fraction.
基金supported by a grant from Union Hospital,Tongji Medical College,Huazhong University of Science and Technology(No.Union-Grant 02.03.2012-03)
文摘This study examined the effect of cholic acid (CA) on cultured cardiac myoeytes (CMs) from neonatal rats with an attempt to explore the possible mechanism of sudden fetal death in intra- hepatic cholestasis of pregnancy (ICP). Inverted microscopy was performed to detect the impact of CA on the beating rates of rat CMs. MTT method was used to study the effect of CA on the viability of CMs. CMs cultured in vitro were incubated with 10 ~maol/L Ca2+-sensitive fluorescence indicator fluo-3/AM. The fluorescence signals of free calcium induced by CA were measured under a laser scanning confocal microscope. The results showed that CA decreased the beating rates of the CMs in a dose-dependent manner. CA could suppress the activities of CMs in a time- and dose-dependent manner. CA increased the concentration of intracellular free calcium in a dose-dependent manner. Our study suggested that CA could inhibit the activity of CMs by causing calcium overload, thereby leading to the sudden fetal death in ICP.
基金Supported by the National Nature Science Foundation of China (30270551) and Military "10.5"Foundation (02M012).
文摘Objective To investigate the effect of peroxisome proliferator-activated receptor-α(PPARα) and PPARγactivators on tumor necrosis factor-α(TNFα) expression in neonatal rat cardiac myocytes. Methods Primary cultures of cardiac myocytes from 1- to 3-day-old Wistar rats were prepared, and myocytes were ex-posed to lipopolysaccharide (LPS) and varying concentrations of PPARαor PPARγactivator (fenofibrate or pioglitazone).RT-PCR and ELISA were used to measure TNFα, PPARα, and PPARγexpression in cultured cardiac myocytes. Transient tr-ansfection of TNFαpromoter with or without nuclear factor-kappaB (NF-κB) binding site to cardiac myocytes was performed. Results Pretreatment of cardiac myocytes with fenofibrate or pioglitazone inhibited LPS-induced TNFαmRNA and protein expression in a dose-dependent manner. However, no significant changes were observed on PPARαor PPARγmRNA expression when cardiac myocytes were pretreated with fenofibrate or pioglitazone. Proportional suppression of TNFαpromoter activity was observed when myocytes was transiently transfected with whole length of TNFαpromoter (-721/+17) after being stimulated with LPS and fenofibrate or pioglitazone, whereas no change of promoter activity was observed with transfection of TNFαreporter construct in deletion of NF-κB binding site (-182/+17). Conclusions PPARαand PPARγactivators may inhibit cardiac TNFαexpression but not accompanied by change of PPARαor PPARγmRNA expression. Therefore PPARαand PPARγactivators appear to play a role in anti-inflammation. The mechanism may partly be involved in suppression of the NF-κB pathway.
基金Supported by the National Natural Science Foundation of China, No.30160028
文摘AIM: To investigate the effects of exogenous unsaturated fatty acids on calcium-activated potassium current [IK(Ca)] in gastric antral circular myocytes of guinea pigs. METHODS: Gastric myocytes were isolated by collagenase from the antral circular layer of guinea pig stomach. The whole-cell patch clamp technique was used to record /K(Ca) in the isolated single smooth muscle cells with or without different concentrations of arachidonic acid (AA), linoleic acid (LA), and oleic acid (OA). RESULTS: AA at concentrations of 2,5 and 10 μmol/L markedly increased IK(Ca) in a dose-dependent manner. LA at concentrations of 5, 10 and 20 μmol/L also enhanced /K(Ca) in a dose-dependent manner. The increasing potency of AA, LA, and oleic acid (OA) on /K(Ca)at the same concentration (10μmol/L) was in the order of AA>LA>OA. AA (10 μmol/L)-induced increase of Ik(ca) was not blocked by H-7 (10 μmol/L), an inhibitor of protein kinase C (PKC), or indomethacin (10 μmol/L), an inhibitor of the cyclooxygenase pathway, and 17-octadecynoic acid (10 μmol/L), an inhibitor of the cytochrome P450 pathway, but weakened by nordihydroguaiaretic acid (10μmol/L), an inhibitor of the lipoxygenase pathway. CONCLUSION: Unsaturated fatty acids markedly increase Ik(Ca), and the enhancing potencies are related to the number of double bonds in the fatty acid chain. The lipoxygenase pathway of unsaturated fatty acid metabolism is involved in the unsaturated fatty acid-induced increase of IK(Ca) in gastric antral circular myocytes of guinea pigs.
文摘The whole-cell patch-clamp technique was employed to obtain information about the voltage-dependence and kinetics of interaction of 7-chlor-benzylte-trahydropalmatine (7-Cl-BTHP) with cardiac sodium channels. 7-Cl-BTHP (30 mol/L) significantly decreased the peak sodium current (from 7. 8±1. 8 nA to 5. 3±1. 4 nA, P<0. 01, n=5), without producing a shift of the current-voltage curve. It shifted the inactivation curves of sodium current to hyperpolarized potentials, and the V(0.5) was shifted from - (82. 5±2. 5) mV to - (95±2.4) mV (P <0. 05, n=4). 7-Cl-BTHP produced a significant use-dependent effect that was proportional to the duration of the voltage step. In addition, 7-Cl-BTHP slowed the recovery of sodium channel from inactivation, which could explain its use-dependent effects on sodium current. The characteristics of 7-Cl-BTHP blockage suggest that this agent binds preferentially to inactivated sodium channels.
基金This research was supported by the National Natural Science Foundation of China (No. 39870334No. 39970273).
文摘Objective To investigate the effects of adenosine (Ado) on myocardiac electrophysiology in simu- lated ischemla and reperfusion in guinea-pig ventricular myocytes. Methods Electrical activity was recorded using standard intracellular microelectrode technique. Right ventricle was superfused with simulated ischemic Tyrode’s so- lution for 15 min, and reperfued with normal Tyrode’s solution for 30 min. Results The results showed Ado had no measurable effects on guinea-pig ventricular myocytes in normal Tyrode’s solution. In the presence of Ado, maximal diastolic potential tended to be more depolarized during ischemia, and action potential (AP) parameters were abbrevi- ated greatly in a concentration-dependent manner. Especially, the concentration of Ado 100 μmol·L-1 had significant effects on AP parameters in ischemic phase [APD30, APD50, and APD90 reduced by (86±8)% versus (65±6)%, (70 ±7)% versus (50±6)%, and (60±6)% versus (42±4)% for control after 15 min, P<0.O5]. During reperfu- sion, AP parameters did not completely return to initial values in presence of Ado. This study illustrated that Ado significantly decreased incidence of arrhythmias induced by ischemia and reperfusion (in presence of Ado 100 μmol· L-1, the incidence of DAD decreased by 17% versus 82% for control during reperfusion). Conclusion Ado has no significant effects on guinea-pig ventricle in normal conditions, abbreviates greatly AP parameters during ischemia with a concentration-dependent manner, and has marked antiarrhythmic effects in ischemia and reperfusion.
文摘Objectives To investigate the anti-apoptotic effects of mesenchymal stem cells (MSCs) on hypoxic injured cardiac myocytes in vitro. Methods MSCs were isolated from bone marrow of Sprague-Dawley (SD) rats, and cardiac myocytes from neonatal rats. The rat cardiac myocytes were co-cultured with MSCs or MSC-conditioned media in anoxia (95% N2 ±5% CO2) for 72 hours. Cell apoptosis was measured by Hoechst 33258 staining. The expression of Bcl-2 and Bax in cardiac myocytes was tested by Western Blot. Results The apoptotic rate was 51.6% ± 2.4% when cardiac myocytes were cultured in continuous hypoxia and was significantly decreased when cardiac myocytes were cocultured with MSCs or MSC-conditioned media ( 15.1% ± 5.4% and 24. 0% ± 4.2% respectively, P 〈 0. 001 ). The decreased expression of Bax in the cardiac myocytes was greatly related to the decreasing of apoptosis, but there was no difference in Bcl-2 expression among these groups. Conclusions Co-cultured MSCs showed significant anti-apoptotic effects on cardiac myocytes in continuous hypoxia. The mechanism may be the interact of cell to cell and paracrine of cytokines which effected the expression of Bax in the cardiac myocytes.
基金This work was supported by the grant from the National Natural Science Foundation of China (grant number. No: 81030002,81170177, 81100215, 81373835).
文摘Objective To explore the effect of allocryptopine (All) on the Late sodium current (INa,Late) of atrial myocytes in spontaneously hyper- tensive rats (SHR). Method The enzyme digestion method was used to separate single atrial myocytes from SHR and Wistar-Kyoto rat (WKY) rats. INa,Late was record by patch-clamp technique and the effect of All on the current was evaluated. Results Comparing with WKY cells, markedly increasing of INa,Late current in SHR myocytes was found from 0.24 ± 0.02 pA/pF of WKY cells to 1.73± 0.04 pA/pF of SHR cells (P 〈 0.01, n = 15). After treament with 30 μmol/L All; the current densities was reduced to 0.92 ± 0.03 pA/pF. The ratio of INa,Late/INa,peak of WKY and SHR were 0.09% ± 0.01% and 0.71% ± 0.02%, INa, Late/INa,peak of SHR was reduced to 0.37% ± 0.02% by 30 μmol/L All (P 〈 0.01, n = 15). We also determined the effect of All on the gating mechanism of the INa,Late in the SHR cells. It was found that All decreased the INa,Late by alleviating the inactivation of the channels and increasing the window current of sodium channel. Conclusion Increased INa,Late in SHR atrial myocytes and the prolonged APD were inhibited by All coming from Chinese herb medicine.
基金Supported by the National Natural Science Foundation of China (No. 30200089 and No. 30500211)
文摘Objectives To analyze and identify differentially expressed phosphoproteins associated with mitochondrial KATP channel opening. Methods: Adult rat ventricular myocytes were isolated, cultured, and identified, and pretreated without or with 100 μmol/L diazoxide for 10 min. Phosphoproteins prepared and enriched from the control and diazoxide-pretreated cells were separated by two-dimensional gel electrophoresis (2-DE) followed by sliver staining. The obtained interesting phosphoproteins were further identified by mass spectrometry. Results. Associated with diazoxide preconditioning, the proteins of chaperonin containing TCP-1 and hypothetical protein XP-346548 were phosphorylated significantly (P〈0. 01), while the 94-kDa glucose-regulated protein, calpactin I heavy chain and ferritin were dephosphorylated markedly (P〈0. 01). Conclusion: These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins in order to respond diazoxide preconditioning, and these phosphorylated protein may mediate the downstream signaling of cardioprotection by mitochondrial KATp channel opening induced by ischemic preconditioning.
文摘Objective To observe the anti-CVB3 ( Coxsackievirus B3 ) effect of sophocarpine (SC) extracted from Sophora flavescens, a traditional Chinese herb in vitro. Methods Cardiomyocytes from the neonatal rat were cultured to establish the viral myocarditis model The cells were divided into four groups: infected group ( infected by CVB3 ) , SC treated group ( added SC 100 μg/mL after viral infection ), SC control group ( added SC 100 μg/mL only), and normal control group. The cytopathic effect (CPE) and the beating frequency of the myocardial cells were observed and the LDH levels in the supernatant were measured at day 2,3, and 5. The cultured myocytes were added different concentrations of SC ( 12. 5 -400 μg/mL ) after infection with CVB3, the CPE was observed and the concentrations of LDH were measured and compared at day 2, 3, and 5. Results In the SC treated group ( 100 μg/mL ) , the cytopathic effect was lighter and the LDH level was lower than the infected group. SC in a concentration of 12. 5 - 300 μg/mL could relieve the CPE and lower the LDH level, while in a higher concentration (400 μ/m ) , it exacerbated the CPE caused by the virus, and the LDH levels were higher than the infected cells. Conclusion SC in certain concentration could protect the cultured rat cardiomyocytes from CVB3 infection.
基金Supported by the Grants from Department of Science and Technology of Jilin Province, China(No.20070726)Bureau of Science and Technology of Changchun City, China(No.2005038).
文摘Reactive oxygen species(ROS) plays a key role in human heart diseases. Glutathione peroxidase(GPX) functions as an antioxidant as it catalyzes the reduction of hydroperoxide. In order to investigate the antioxidant effect of human selenium-containing single-chain Fv(Se-scFv-B3), a new mimic of GPX, a model system of hydrogen peroxide(H202)-induced rat cardiac myocyte damage was established. The cardiac myocyte damage was characte- rized in terms of cell viability, lipid peroxidation, cell membrane integrity, and intracellular H202 level. The Se-scFv-B3 significantly reduced H2O2-induced cell damage as shown by the increase of cell viability, the decline of malondialdehyde(MDA) production, lactate dehydrogenase(LDH) release, and intracellular H2O2 level. So Se-scFv-B3 may have a great potential in the treatment of human heart diseases induced by ROS.
基金the National Natural Science Foundation of China(30901058 and 30972157)the National Basic Research Program of China(2009CB941601)+2 种基金the Natural Science Foundation of Guangdong Province of China(S2012010010176 and S2012020011048)the Special Fund for Agro-Scientific Research in the Public Interest,China(201003011)the Specialized Research Fund for the Doctoral Program of Higher Education of China(20094404120012)
文摘Bone marrow mesenchymal stem cells (BMSCs) could differentiate into various cell types including adipocytes and myocytes, which had important scientific significance not only in the field of tissue regeneration, but also in the field of agricultural science. In an attempt to exhibit the characterization and differentiation into adipocytes and myocytes of porcine BMSCs, we isolated and purified porcine BMSCs by red blood cell lysis method and percoll gradient centrifugation. The purified cells presented a stretched fibroblast-like phenotype when adhered to the culture plate. The results of flow cytometry analysis and immunofluorescence staining demonstrated that the isolated cells were positive for mesenchymal surface markers CD29, CD44 and negative for hematopoietic markers CD45 and the adhesion molecules CD31. Cells were induced to differentiate into adipocytes with adipogenic medium containing insulin, dexamethasone, oleate and octanoate. Oil Red O staining demonstrated that the porcine BMSCs successfully differentiated to adipocytes. Moreover, the findings of real-time PCR and Western blotting indicated that the induced cells expressed adipogenic marker genes (PPAR-y, C/EBP-c~, perilipin, aP2) mRNA or proteins (PPAR-3,, perilipin, aP2). On the other hand, porcine BMSCs were induced into myoctyes with myogenic medium supplemented with 5-azacytidine, basic fibroblast growth factor, chick embryo extract and horse serum. Morphological observation by hochest 33342 staining showed that the induced cells presented as multi-nucleus muscular tube structure. And myogenic marker genes (Myf5, desmin) mRNA or proteins (MyfS, MyoD, myogenin, desmin) were found in the induced cells. In addition, the results of immunofluorescence staining revealed that myogenic marker (Myf5, MyoD, myogenin, desmin, S-MyHC) proteins was positive in the induced cells. Above all, these results suggested that the isolated porcine BMSCs were not only consistent with the characterization of mesenchymal stem cells, but also exhibited the multipotential capacity to form adipocytes and myocytes, which provided the basis to investigate the regulation mechanism involved in the selective differentiation of porcine BMSCs.
基金Supported by the National Natural Science Foundation of China(Nos.30870540,30970633)
文摘As one of the most important antioxidant enzymes, glutathione peroxidase(GPX) protects cells and tissues from oxidative damage, and plays an important role in cardiovascular and cerebrovascular injuries induced by oxida- tive stress. The antioxidant effect of selenium-containing glutathione S-transferase(Se-GST), a mimic of GPX was investigated on rat cardiomyocytes. To explore the protection function of Se-GST in hydrogen peroxide(H202) chal- lenged rat cardiomyocytes, we examined malondialdehyde(MDA), lactate dehydrogenase(LDH), superoxide dismu- tase(SOD) and cell apoptosis. The results demonstrate exposure of rat cardiomyocytes to H202 for 6 and 12 h induced the significant increases of MDA, LDH and apoptosis rate of cardiomyocytes, but pretreatment of rat cardiomyocytes with Se-GST at 0.0005 or 0.001 unit/mL prevents oxidative stress induced by H202 with the decreases of cell apopto- sis. All the results hint Se-GST has antioxidant activity for oxidative stress challenged rat cardiomyocvtes.
文摘Objective:To observe the protective effects of erythropoietin (EPO) pretreatment on cardiac myocyte with hypoxia/reoxygenation (H/R) injury and the role of NF-κBin this effects. Methods:After the H/R model of cardiac myocytes of neonatal rats was established, the cultured cardiac myocytes were divided into 4 groups, including EPO pretreatment group ( EPO 10 U/ml 24 h before H/R), EPO pretreatment + PDTC group(EPO 10 U/ml and PDTC 5 μg/ml 24 h before H/R), PDTC group (PDTC 5 μg /ml 24 h before H/R) and eomrolgroup. Before and after the H/R, assay of LDH concentration in the culture medium, the survival rate of the myocytes tested by MTT chromatometry and the apoptosis by flow cytometry were undertaken. Activation of NF-κB was determined by EMSA before and after H/R. Results:EPO pretreatment markedly reduced the LDH concentration in the medium, elevated the survival rate of myocytes and inhibited the apoptosis after H/R. Addition of PDTC during the pretreatment abol- ished the protective effects of EPO pretreatment. NF-κB was markedly activated during EPO pretreatment and PDTCinhibited the activation. However, after H/R, the activity of NF-κB in myocytes with EPO pretreatment was significantly inhibited compared to the other myocytes. Conclusion:NF-κB is significantly activated during EPO pretreatment, but is inhibited after H/R, which is correlated with the protective effects of EPO pretreatment on cardiac myocytes with H/R. This phenomenon can be explained as the negative feedback mechanism of the activation of NF-κB.
文摘Objectives To investigate the effects of β2-adrenergic antagonist on cytosolic Ca^2 + ([Ca^2+ ]i) in ventricular myocytes from infarcted rat heart. Methods A ligature was placed around left anterior descending coronary artery of rat hearts. Rats in the control group were sham-operated. Cardiomyocytes were dissociated at two, four, eight weeks after myocardial infarction (MI) and [Ca^2+]i was measured via fura-2 fluorescence. The response of cardiomyocytes to isoproterenol in presence or absence of betal-adrenergic antagonist atenolol, beta2-adrenergic antagonist ICI118, 551 or non-selective β1, 2- adrenergic antagonists propranolol was examined. Results The followings were found that ICI 118, 551 had no significant effects on the rise of [Ca^2+]i induced by isoproterenol in normal ventricular myocytes (P 〉 0.05), ICI118, 551 only significantly attenuated the rise of [Ca^2+]i induced by isoproterenol at four weeks and eight weeks after MI (24.5%±5.7% vs 57.8% ± 13.2%, P〈 0.01; 12.2%±7.9% vs 44.6%±11.3%, P〈 0.01). Atenolol had suppressive effects only in the control group and the post-MI group of two weeks (P 〈 0.05), and propranolol had suppressive effects in the control and all the three post-MI groups (P 〈 0.01). Conclusions Beta2-adrenergic antagonist ICI118, 551 may exert negative effects on Ca^2+ overload initiated by sympathetic stimulation after MI.
文摘Objective To study the effects of simvastatin on the hypertrophy of cultured rat cardiac myocytes induced by serum and the role ofphosphatase and tensin homolog deleted on chromosome ten (PTEN) in the signal pathway. Methods Cultured neonatal Sprague- Dawley (SD) rat cardiac myocytes were treated with 15% fetal bovine serum, or without serum, or different consentrations of simvastatin. Image analysis system was used to measure the cardiac myocytes surface area. Protein synthesis of myocytes was measured via [3H]-leucine incorporation method. The expression level of atrial natriuretic peptide (ANP) mRNA in myocytes was determined with reverse transcription polymerase chain reaction (RT-PCR). The mRNA and protein expression levels of PTEN in cardiac myocytes were investigated with RT-PCR and Western blot respectively. Results At 24 hours, cardiac myocytes surface area was significantly higher in 15% serum group (1611.16± 160.75 lam2) than in serum-free group (538.04±118.60 ±tm2, P〈0.01). Simvastatin decreased the cell surface area in a concentration dependent manner. The cell surface area in 10-5 and 10-6 mol/L simvastatin groups were 799.84+ 167.70 ±tm2 and 1076.88± 199.28 um2 respectively, which were both significantly lower than that in 15% fetal bovine serum group (P〈0.01). Incorporation rate of [3H]-leucine was significantly higher in 15% fetal bovine serum group (2360± 106cpm/well) than that in serum-free group (1305±92 cpm/well, P〈0.01). Incorporation rate of [3H]-leucine in 10.5 and 10.6 mol/L simvastatin groups were 1707±101 clam/well and 1962±125 cpm/well respectively, which were both lower than that in serum group (P〈0.01). With the increase of simvastatin concentration, the expression level ofANP mRNA in cardiac myocytes was decreased gradually, which were 0.29±0.03 and 0.40-±0.03 respectively in 10.5 and 10-6 mol/L simvastatin groups, and significantly lower than that in serum group(0.60-±.03, P〈0.01). Simvastatin increased the expressions of PTEN mRNA and protein in cardiac myocytes in a concentration dependent manner. PTEN mRNA expression level in 10-7, 10-6and 105mol/L simvastatin groups were 0.38±0.03, 0.83±0.04 and 0.85±0.05, respectively, which were all higher than that in 15% fetal bovine serum group (0.29±0.04, P〈0.05). Similarly, PTEN protein level in 10-7, 10-6 and 10.5 mol/L simvastatin groups (39.25±3.41, 46.35±1.78 and 47.22±2.39 respectively) were also significantly higher than that in 15% fetal bovine serum group (32.21±4.06, P〈0.05). Conclusion Simvastatin can inhibit the hypertrophy of cultured rat cardiac myoeytes induced by serum, and the increase of expression level of PTEN might be involved in the mechanism (J Geriatr Cardio12010; 7:47-51).