Effect of carvedilol on store overload-induced Ca2+ release in rat pacing cardiomyocytes

AIM：To explore the effect of carvedilol on store overload-induced Ca2+ release (SOICR) in pacing cardiomyocytes. METHODS：Single rat cardiomyocyte with rapid pacing was perfused with isoprenaline and caffeine to induce calcium overload. Spontaneous calcium releases through sarcoplamic reticulum calcium release channel (ryanodine receptor 2, RyR2) were investigated by the method of fluorescence imaging. The cardiomyocytes were divided into control (DMSO) group, carvedilol group, metoprolol group, phentolamine group and nifedipine group. RESULTS：In control group, the incidence of SOICR in cardiomyocytes was significantly increased under the condition of calcium overload by perfusing with isoprenaline and caffeine in addition to the enhancement of calcium transient. The incidence of SOICR in carvedilol group was significantly lower than that in control group at the pacing frequency of 1 Hz to 4 Hz (2.00%, 6.00%, 10.00% and 16.00% vs 43.59%, 74.36%, 87.18% and 89.71%, respectively, P<0.01). The inhibitory effect of carvedilol was not significantly different at variant pacing frequency (P>0.05). The incidences of SOICR in metoprolol group, phentolamine group and nifedipine group had no significant difference compared with control group (P>0.05). The amplitude of calcium transient and caffeine peaking value of pacing cardiomyocytes had no significant difference among different groups (P>0.05). CONCLUSION：Carvedilol effectively suppresses SOICR in pacing cardiomyocytes due to its direct inhibition on the spontaneous opening of the cardiac RyR2 channel rather than the α1, β1 receptor or L-type calcium channel blockade.     

Effects of extracellular potassium on expression of HERG gene nonsense mutant L539fs/47

AIM: To study the effects of extracellular potassium on the protein expression of wild- type HERG and its mutant L539fs/47. METHODS: Wild-type HERG (WT) or its mutant HERG-L539fs/47 (MT) were transfected into HEK293 cells for 36 h. The cells were incubated in different media containing 0.8, 4.3 or 10 mmol/L potassium. After 6 h of incubation, the protein expression of HERG was detected by flow cytometry.After 12 h of incubation, the localization and quantity of the proteins were detected by laser confocal imaging and Western blot. RESULTS: Different from the retention of mutant protein in cytoplasm, wild-type HERG protein was mainly distributed in the cell membrane. The 2 proteins both increased with the changes of extracellular potassium. Flow cytometry showed that the fluorescence in the 2 groups both increased with the changes of extracellular potassium (P < 0.01). The fluorescence in WT group was significantly higher than that in MT group (P < 0.01). Western blot showed that mutant HERG protein included only one 60 kD band, different from the 135 kD and 155 kD bands in wild-type HERG, which were affected by the changes of extracellular potassium (P < 0.05). CONCLUSION: The retention of HERG mutant L539fs/47 protein in the cytoplasm is more than wild-type HERG. Chronic high extracellular potassium keeps the stability of wild-type and mutant HERG proteins on the cell membrane. Chronic low potassium reduces the expression of HERG channel proteins in a time-dependent manner.     

Effects of carvedilol on metabolic mechanism of endogenous NOS inhibitor in human umbilical vein endothelial cells treated with oxidized free radical

AIM: To observe the effects of oxidized free radical (OFR) on dimethylarginine dimethylaminohydrolase (DDAH) activity and concentration in human umbilical vein endothelial cells (HUVECs), and to investigate the metabolic mechanism of endogenous NOS inhibitor and the role of carvedilol. METHODS: HUVECs of 3-6th passage, cultured with modified Jaffes method, were divided into three groups: (1) cells cultured with equivalent DMEM medium as control; (2) OFR intervention groups, 0.01 mmol/L, or 0.1 mmol/L OFR was added respectively; (3) drug intervention groups: 0.1 mmol/L OFR plus 10 μmol/L carvedilol. ADMA, nitric oxide (NO), endothim (ET), L-citrulline concentrations and the activity of NOS in conditioned medium were measured after 24 h exposure. ADMA concentration in the conditioned medium was determined by high-performance liquid chromatography. Western blotting was performed to evaluate DDAH expression. RESULTS: Compared with control group, ADMA and ET concentrations were increased, while the level of NO and the activity of NOS decreased and relevant to the concentrations of OFR. We assayed DDAH activity by determining L-citrulline formation from ADMA. The concentration of L-citrulline was decreased, while the DDAH expression had no obvious change. With the role of carvedilol, ADMA, ET concentrations were decreased, while the level of NO, L-citrulline and the activity of NOS increased. CONCLUSION: Endothelial dysfunction induced by OFR is associated with the increase in ADMA concentration and reduction of DDAH activity, but not DDAH expression. Carvedilol promotes the degradation of ADMA through increasing activity of DDAH and improving endothelium function.     

Effects of carvedilol,cilazapril and their combination on left ventricular remodeling after acute myocardial infarction in rats

AIM:To compare the effects of carvedilol, cilazapril and their combination on left ventricular remodeling(LVRM) after acute myocardial infarction(AMI) in rats. METHODS: Twenty-four hours after AMI operation, 100 surviving rats were randomly assigned to: ①AMI control(n= 25), ②AMI+carvedilol(1 mg·kg^-1 ·d^-1, n= 25)(C1), ③AMI+cilazapril(1 mg·kg^-1 ·d^-1, n= 25)(Z1), and ④ AMI+combination(n= 25) groups. Sham-operated group(n= 17) were selected randomly. After 4 weeks of therapy with the drugs gastric gavage, hemodynamic and pathological studies were performed. RESULTS: There were no significant differences in MI size among the four AMI groups(all P> 0.05) Left ventricular(LV) end diastolic pressure(LVEDP), volume(LVV), weight(LVW) and septal thickness(STh) were all higher and left ventricular pressure maximal rate of rise and fall(±d p /d t) were lower(all P< 0.01) in AMI group than sham-operated group. The LVEDP, LVV, LVW and STh were all lower and ±dp /dt were higher in Z1, C1, and combination groups than those in AMI group(P< 0.05, P< 0.01), with LVEDP and STh were more lower in the combination group than in the two monotherapy group(P< 0.05, P< 0.01), but there were no significant differences in other variables among the three therapy groups. CONCLUSION: Carvedilol, cilazapril and their combination all can prevent from LVRM after AMI in rats, improve hemodynamics and LV function, with the combination superior.     

喷施钙肥对梨果实品质和石细胞代谢的影响

石细胞是影响梨果实品质的关键因素之一,钙与石细胞次生壁形成过程关系密切。以南果梨为试材,于花后不同时期喷施钙肥,研究了不同浓度的氯化钙及糖醇螯合钙对其果实品质及石细胞代谢的影响。结果表明,与对照相比,不同钙肥处理对南果梨果实品质均有所改善,同时显著提高果实钙含量,并对石细胞形成有不同程度的抑制作用。其中,盛花期后20d喷施5.0g/L氯化钙可显著增加南果梨单果重、横纵径和总钙含量,并减少石细胞积累,效果最佳。进一步研究发现,氯化钙处理降低了木质素合成相关基因PuC3H、Pu CAD、PuPOD、PuLAC的表达水平。因此,钙处理可通过抑制木质素途径下游基因的表达来减少石细胞的积累,是提高南果梨果实品质的有效措施。     

Effects of resveratrol on the L-type calcium current by protein kinase G pathway in isolated guinea pig ventricular myocytes

AIM: To investigate the effects of resveratrol on the L-type calcium current in isolated guinea pig ventricular myocytes. METHODS: The whole cell patch clamp method was used. RESULTS: （1）Resveratrol (1, 50, 100 μmol/L) reduced the I_Ca-L by 18.31%±3.15%, 56.20%±2.50% and 84.51%±4.01% in a concentration-dependent manner (n=5, P<0.05). But it has no change on I-V shape of I_Ca-L. (2) 8Br-cGMP (100 μmol/L), an activator of protein kinase G(PKG), deduced the density of I_Ca-L by 10.50%±1.11%. Applying resveratrol and 8Br-cGMP simultaneously decreased the I_Ca-L significantly by 87.58%±3.49％ (n=6, P<0.05). (3) 5 μmol/L H8, a PKG inhibitor, inhibited the decrease in I_Ca-L caused by resveratrol. CONCLUSION: Resveratrol inhibits I_Ca-L in guinea pig ventricular myocytes, and this inhibitory effect involves the PKG pathway.     

Adeno-associated virus type 9-mediated p65 silencing inhibits angiotensin II-induced apoptosis of H9c2 cells

AIM: To study the effect of p65 gene silencing by adeno-associated virus type 9 (AAV9)-mediated RNA interference on angiotensin Ⅱ (Ang Ⅱ; 10^-6 mol/L for 24 h)-induced apoptosis of rat ventricular H9c2 myocytes, and to elucidate the possible mechanism. METHODS: The H9c2 cells were transfected with rAAV9-eGFP and rAAV9-eGFP-NF-κB p65-siRNA at multiplicity of infection (MOI)=4×10⁶ vg/cell. eGFP expression in the cells was observed under an inverted fluorescence microscope, and the percentage of eGFP positive cells was determined by flow cytometry. The expression of p65 was determined by Western blot. CCK-8 assay was used to measured the viability of transfected H9c2 cells. The apoptosis of the cells transfected with the virus and with Ang Ⅱ stimulation was analyzed by flow cytometry. RESULTS: The cells began to exhibit eGFP expression on the 2nd day after transfection. The fluorescence intensity was increased over the time of transfection. eGFP expression reached the maximum on the 5th day, and the transfection efficiency was (52.7±1.9)% at this time point. Compared with blank control group, no significant effect of AAV9 on the viability of H9c2 cells was observed. In resting state, p65 in the H9c2 cells had a certain activity. After Ang Ⅱ stimulation, the activity of p65 was obviously increased, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited the expression of p65. The apoptosis of H9c2 cells in Ang Ⅱ stimulation group was significantly higher than that in blank control group, while transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibited apoptosis of H9c2 cells. CONCLUSION: Transfection of rAAV9-eGFP-NF-κB p65-siRNA effectively inhibits the expression of p65 gene of NF-κB pathway in the H9c2 cells without causing cell growth inhibition, and reduces the apoptosis induced by Ang Ⅱ.     

Effects of orexin-A on food intake and spontaneous physical activity in obesity-resistant rats and high-fat diet-induced obese rats

AIM: To investigate the differences of food intake and spontaneous physical activity (SPA) among obesity-resistant (OR) rats, diet-induced obese (DIO) rats and normal Sprague-Dawley (SD) rats and the role of orexin-A in these processes. METHODS: The rostral lateral hypothalamic area (rLHa) catheter was implanted into the OIO, OR, and SD rats. Orexin A at doses of 0, 31.25, 62.5, 125 and 250 pmol was injected through the catheter. The SPA and food intake were measured and recorded for 2 h after injection. The mRNA expression of prepro-orexin, orexin-A receptor (OX1R) and orexin-B receptor (OX2R) in the rLHa and hypothalamus of OR, DIO and SD rats was detected by real-time PCR. The protein expression of OX1R and OX2R in the hypothalamus and rLHa of the rats was measured by radioimmunoassay. RESULTS: A small-dose injection of orexin-A into rLHa significantly increased the food intake in all the rats. Orexin A-induced SPA had significant differences, showing that the OR and SD rats had the higher motion than the DIO rats. The mRNA and protein levels of OX1R and OX2R in the rLHa of OR rats were significantly higher than those in DIO and SD rats. CONCLUSION: Hypothalamic orexin-A participates in the regulation of energy metabolism in obese and normal rats, in which the regulatory effect on OR rats is the best.     

猕猴桃果实成熟前补钙对果实含钙量的影响

猕猴桃果实成熟前用喷叶法和喷果法补钙，可显著提高果实中的钙含量。喷叶较喷果效果好。用Ca(NO3)2喷叶，比用CaCl2处理效果好，差异显著。而果实对CaCl2和Ca(NO3)2的吸收选择性差异不显著。经补钙处理的果实采后进行贮藏效果观察发现：用Ca(NO3)2喷叶处理的果实贮藏性较好，用其喷果处理次之；用CaCl2喷果和喷叶处理的果实贮至28天软果率均达到90％。     

Effect of SCF and G-CSF pretreatment on the proliferation and the differentiation of bone mesenchymal stem cells

AIM: To investigate the effect of pretreatment of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) on the proliferation and the differentiation of mesenchymal stem cells (MSCs) into cardiomyogenic cells. METHODS: The MSCs, isolated primarily from bone marrow, and purified by passage culture, were obtained from the adult rats of four groups: the rats were pretreated by 5 daily injections of SCF; the rats were pretreated with G-CSF; the rats were pretreated with SCF and G-CSF; the rats were treated without any intervention. The 4th passage of MSCs was labeled by DAPI and cellular cycle analysis was conducted by flow cytometry before co-culture. The neonatal rat cardiomyocytes cultured for 3 days were co-cultured with DAPI-MSCs. The percentage of the differentiation of MSCs into cardiomyogenic cells during the five co-culture days was analyzed. The morphologic changes of MSCs and the proteins expression of cardiac myosin heavy chain (MHC) and troponin T (TnT) were recorded respectively with digital microscope camera system and immunofluorescence technique. The percentage of the differentiation of MSCs into cardiomyogenic cells was also calculated. RESULTS: The percentage of MSCs in G₀/G₁ phase in SCF/G-CSF group was significantly lower than that in SCF group, G-CSF group and the control group. The percentage of MHC protein-positive MSCs in SCF/G-CSF group was markedly higher than that in SCF group, G-CSF group and the control group, and that in SCF group and G-CSF group was significantly higher than control group. The percentage of TnT protein-positive MSCs in SCF/G-CSF group, SCF group and G-CSF group was significantly higher than that in control group.CONCLUSION: SCF and G-CSF show the ability to stimulate the proliferation of MSCs and induce MSCs to differentiate into cardiomyocytes. The combination of using SCF and G-CSF is more effective than using only SCF or G-CSF.     

Effects of nitric oxide on spontaneous action potentials of guinea-pig left ventricular outflow tract under the conditions of ischemia/reperfusion

AIM: To study the electrophysiological effects of nitric oxide (NO) on the pacemaker cells in guinea-pig left ventricular outflow tract under the condition of ischemia/reperfusion (I/R). METHODS: The spontaneous slow action potentials of guinea-pig left ventricular outflow tract were recorded by conventional intracellular microelectrode technique. The effects of NO donor sodium nitroprusside (SNP) on the spontaneous slow action potentials under normal or I/R condition were investigated. RESULTS: SNP at concentrations of 1, 10 and 100 μmol/L but not 1000 μmol/L significantly increased velocity of diastolic depolarization (VDD) and rate of pacemaker firing (RPF), SNP at concentrations of 1, 10, 100 and 1 000 μmol/L notably increased maximal diastolic potential (MDP), amplitude of action potential (APA) and maximal rate of depolarization (V_max), shortened 50% and 90% of the duration (APD₅₀ and APD₉₀). In ischemia 10 min group, VDD and RPF were significantly decreased, APA and V_max were notably increased, and APD₅₀ and APD₉₀ were markedly lengthened compared with control group. In reperfusion 10 min group, VDD and RPF were significantly increased, MDP and APA were notably decreased, and APD₅₀ and APD₉₀ were markedly shortened compared with I 10 min group. In reperfusin 10 min group, the pacemaker activity was always irregular. In reperfusion 10 min group, the parameters of spontaneous slow action potentials restored to the levels of control group except for VDD and V_max. In 1, 10 and 100 μmol/L SNP+R groups, VDD and RPF were significantly increased than in ischemia 10 min group. In 1, 10, 100 and 1 000 μmol/L SNP+R groups, APA, APD₅₀ and APD₉₀ were restored to the levels of control group. CONCLUSION: SNP significantly increases the spontaneous activity of left ventricular outflow tract, and relieves the effects of I/R on the spontaneous slow action potential markedly.     

Effects of thyroid hormone on myocardial T-type calcium channels at mRNA and protein levels in rats

AIM: To observe the effect of thyroxine on the expression of T-type calcium channels Cav3.1, Cav3.2 and Cav3.3 in rat myocardium, and to explore the possible biological mechanism between the changes of the expression of T-type calcium channels and the arrhythmia in hyperthyroid heart disease. METHODS: Healthy SD rats (n=20) were randomly divided into normal control group (n=10) and hyperthyroid heart disease group (n=10). The animal model was established by intraperitoneal injection of levothyroxine for 35 d. The contents of T3 and T4 in serum, the heart-to-body weight ratio, the diameter of cardiac myocytes and electrocardiograph were measured to evaluate hyperthyroid heart disease. Moreover, the mRNA and protein expression levels of T-type calcium channels in the myocardium were measured by RT-PCR, immunohistochemistry and Western blot. RESULTS: After intraperitoneal injection of levothyroxine for 35 d, compared with the normal control group, the serum contents of T3 and T4, the heart-to-body weight ratio and the diameter of cardiac myocytes were significantly increased in hyperthyroid heart disease group (P<0.05), and arrhythmia occurred in hyperthyroid heart disease group. By immunohistochemistry and Western blot, the protein expression of Cav3.1 increased significantly (P<0.05), while the protein expression of Cav3.2 decreased significantly (P<0.01). However, no change of the Cav3.3 protein was observed. The results of RT-PCR were the same as immunohistochemistry and Western blot. CONCLUSION: Thyroxine promotes the expression of Cav3.1 in the myocardium but inhibits the expression of Cav3.2 at mRNA and protein levels, which might be involved in arrhythmia in hyperthyroid heart disease.     

Effects of night-time humidity and nutrient solution concentration on the calcium content of tomato fruit

Calcium intake into tomato fruits was greater when nights were humid rather than dry and nutrient solutions dilute rather than concentrated.The concentration of calcium in the wall tissue of the distal segment of fruits damaged by blossom-end rot was 0.03% of dry matter, but was 2- to 3-fold greater in the most favourable conditions of humidity and solution concentration, when fruits were undamaged.Adding extra calcium to the nutrient solution increased the calcium concentration in the proximal, but not in the middle or distal, segments of the fruit.The results support the hypothesis that a positive root pressure at night promotes transport of calcium into tissues and organs that have restricted transpiration.     

Effects of cardiomyopeptidin on sodium current in ventricular myocytes of guinea pigs

AIM: To determine the effect of cardiomyopeptidin on sodium current (I_Na) in ventricular myocytes of guinea pigs and to explore the mechanism of cardiomyopeptidin action at the ionic channel level. METHODS: Single ventricular myocytes of guinea pigs were obtained by enzymatic dissociation method. The whole-cell patch-clamp recording technique was used to record the change of I_Na. RESULTS: Cardiomyopeptidin (1, 5, 10, 50, 100 and 500 mg/L) decreased I_Na in a dose-dependent manner. The inhibition rates were (0±1)%, (6±2)%, (10±2)%, (15±1)%, (22±9)% and (30±6)%, respectively. The time to peak (TTP) was delayed from (2.8±0.7) ms to (3.0±0.8) ms (P<0.05) by cardiomyopeptidin (50 mg/L). In the presence of cardiomyopeptidin (50 mg/L), the current density-voltage curve of I_Na was shifted and without change of its active potential, peak potential, reversal potential, and the shape of the curve. The steady activation curve, the steady inactivation curve and the steady inactivation recovery curve of I_Na were not affected. CONCLUSION: Cardiomyopeptidin inhibits the I_Na in guinea pig ventricular myocytes, which may be one of the mechanisms of its antiarrhythmic effect.     

Protective effects and mechanisms of losartan on apoptosis of H9c2 cells induced by β-adrenergic stimulation in vitro

AIM: To investigate the protective effect of losartan (Los) on apoptosis of H9c2 cells induced by isoprenaline (ISO), and to discover its related mechanism. METHODS: H9c2 cells cultured on plastic plates were divided into control, ISO, ISO+Los, ISO+Los+LY294002 and DMSO groups. Cell apoptosis was evaluated by flow cytometery and agarose gel electrophoresis. The mRNA levels of bax, bcl-2 and caspase-9 were detected by RT-PCR and the expressions of phosphorylated and total Akt (p-Akt and t-Akt) were assessed by Western blotting. The cAMP was measured by radioimmunoassay. RESULTS: ISO at concentration of 10 μmol/L induced apoptosis of H9c2 with an increase in bax/bcl-2, caspase-9 and cAMP. Addition of 10 μmol/L losartan inhibited apoptosis obviously with a decrease in bax/bcl-2, caspase-9 and cAMP. A significant increase in p-Akt was observed, and its protein level was elevated. LY294002 at concentration of 1 μmol/L abolished the protective effects of losartan on ISO-induced apoptosis in H9c2 cells. CONCLUSION: ISO might induce H9c2 cell apoptosis through stimulation of β-adrenergic receptor (β-AR). Los inhibits downstream signaling of β-AR, and promotes the activation of Akt. Subsequently it might attenuate the apoptosis induced by β-adrenergic stimulation of ISO.     

Influence of captopril on intracellular free calcium concentration and involved ion channels mechanisms in cardiac myocytes of the neonatal rat undergone anoxia-reoxygenation injury

AIM:To observe the influence of captopril on intracellular free calcium concentration (［Ca2+］ i) and the involved ion channels mechanisms in cardiac myocytes of the neonatal rat undergone anoxia-reoxygenation injury.METHODS:The anoxia-reoxygenation model in cultured neonatal rat ventricular myocytes was established.Groups were divided into ① normal;② anoxia-reoxygenation;③anoxia-preconditioning (5 min anoxia+5 min reoxygenation);④ captopril preconditioning.Flou-3 /AM loading and flow cytometry technique were used to observe the ［Ca2+］i,and whole-cell patch clamp technique was used to record the L-type calcium current and Na+/Ca2+ exchange current.RESULTS:① Compared to normal group,［Ca2+］i in anoxia -reoxygenation group was increased significantly (789.42±9.05 vs 414.08±37.40,P<0.01),L-type calcium current density was decreased (P<0.01),the current-voltage curve was moved up,the inactivation curve was moved left and Na+/Ca2+ exchange current was increased in anoxia-deoxygenating.② Compared to anoxia-reoxygenation group,anoxia and captopril preconditioning resulted in a significant decrease in ［Ca2+］i (593.84±5.06,507.08±31.89 vs 789.42±9.05,P<0.01),and a significant increase in L-type calcium current density (P<0.01),the current-voltage curve was moved down,the inactivation curve was moved right and Na+/Ca2+ exchange current was decreased ③ Compared to normal oxygen condition,the anoxia and captopril precondition resulted in a lightly increase in ［Ca2+］i (507.08±31.89 vs 414.08±37.40,P<0.05) and Na+/Ca2+ exchange current.④ Compared to anoxia-preconditioning group,captopril-preconditioning resulted in no significant difference in all the markers mentioned above.CONCLUSIONS:The anoxia-reoxygenation injury in cardiac myocytes results in ［Ca2+］i abnormal increase and calcium overload by increasing Na+/Ca2+ exchange current.Late preconditioning in cardiac myocytes is triggered by transient and repeated anoxia and captopril,which slightly increases Na+/Ca2+ exchange current and ［Ca2+］i and restraines the abnormal increasing of Na+/Ca2+ exchange current and calcium overload induced by subsequenced anoxia-reoxygenation injury,so it plays an delayed protective role in cardiac myocytes.L-typed calcium passage is not involved in calcium overloaded and late preconditioning of calcium in myocytes during reperfusion.     

Effects of calcium channel blockers on nicotinic acetylcholine receptor current in rat chromaffin cells

AIM: To study the effects of calcium channel blockers (CCB) on nicotinic acetylcholine receptor current (I_NIC) in rat adrenal medullar chromaffin cells (RAMCs). METHODS: By using the whole-cell clamp-patch technique, we have investigated the effects of nifedipine(NIF)、ω-conotoxin GVIA and ω-agatoxin IVA on I_NIC induced by nicotine(NIC) before and after RAMCs perfusion. RESULTS: After perfusing RAMCs for 5 min, different kinds of calcium channel blockers at different concentration showed significant inhibitory effects on I_NIC induced by 50 μmol/L NIC. The peak inhibition rates of 10 μmol/L NIF、400 nmol/L ω-conotoxin GVIA and 100 nmol/L ω-agatoxin IVA were (61.7±5.1)%,(29.3±7.4)% and (17.6±7.5)%, respectively. CONCLUSION: The acute effects of different kinds of CCBs on RAMC were that they obviously inhibited I_NIC induced by NIC. These results suggest that CCBs may inhibit catecholamine secretion by directly blocking nicotinic acetylcholine receptor channel.     

Effects of CYP2J3/EETs on myocardial apoptosis under the condition of hypoxic postconditioning

AIM: To investigate the effects and underlying mechanism of endogenous cytochrome P450 2J3/epoxyeicosatrienoic acids (CYP2J3/EETs) system on myocardial apoptosis under the condition of hypoxic postconditioning (HPost).METHODS: Primary myocardial cells from neonatal Wistar rats (12 h-24 h) were cultured and divided into 7 groups as follows: control group, hypoxia/reoxygenation (H/R) group, HPost group, CYP2J3 transfection group, empty vector group, 6-(2-propargyloxyphenyl)hexanoic acid (PPOH, an inhibitor of CYP2J3) group and dimethyl sulfoxide (DMSO, as solvent) group. The H/R treatment was conducted in all the groups except control group. The cell viability was tested by MTT method. The concentration of 11,12-EET in the cell culture medium was measured by high-perfor-mance liquid chromatography (HPLC). The expression of caspase-3 was detected by Western blotting and its activity was determined by caspase-3 activity assay kit.RESULTS: Compared with H/R group, the cell viability and the 11,12-EET concentration were significantly elevated in HPost group (P<0.01). The two indexes in CYP2J3 transfection group were higher than those in HPost group (P<0.01), but they were lowered in PPOH group than those in HPost group (P<0.01). The comparisons of the expression and activity of caspase-3 among groups were as follows: H/R group > HPost group (P<0.01), PPOH group > HPost group (P<0.01) and empty vector group > CYP2J3 transfection group (P<0.05). Additionally, the caspase-3 expression in CYP2J3 transfection group was lower than that in HPost group (P<0.01).CONCLUSION: The CYP2J3/EETs system protects myocardial cells through inhibiting caspase-3-induced apoptosis.     

Effects of T-cadherin on hypoxia/reoxygenation-induced apoptosis of cardiomyocytes from neonatal rats

AIM: To investigate the mechanism of cardiomyocyte apoptosis induced by hypoxia/reoxygenation (H/R) by silencing a new adiponectin receptor T-cadherin through adenovirus-mediated RNA interference. METHODS: The primary cardiomyocytes were isolated from neonatal rats and cultured for 72 h. The cardiomyocytes were randomly divided into control group, H/R group, APN+H/R group, Ad-T-cadherin-siRNA+APN+H/R group and Ad-HK (adenovirus negative control)+APN+H/R group. The transfection ability and efficiency were examined. The expression of T-cadherin at mRNA and protein levels was detected by RT-PCR and Western blotting. The apoptotic rate was analyzed by flow cytometry and TUNEL. RESULTS: High purity of neonatal rat cardiomyocytes was obtained by primary culture. After 48 h, over 90% of myocardiocytes were infected at MOI=100. The transfected myocardiocytes showed a low expression level of T-cadherin under normal physiological condition. Compared with APN+H/R group, the cell apoptotic rate significantly increased in Ad-T-cadherin-siRNA+APN+H/R group (P<0.05). Compared with H/R group, the difference was not statistically significant (P>0.05). CONCLUSION: Ad-T-cadherin-siRNA effectively infects myocardial cells in vitro and successfully reduces the expression of T-cadherin in myocardial cells. The inhibitory effect of adiponectin on H/R-induced cardiomyocyte apoptosis is attenuated by decreasing the expression of T-cadherin.