Effect of interleukin-2 on intracellular calcium levels in rat ventricular myocytes during anoxia and reoxygenation

AIM: To investigate the effect of interleukin-2(IL-2) on the intracellular calcium in electrically stimulated adult rat ventricular myocytes during anoxia and reoxygenation. METHODS: The isolated cardiac ventricular myocytes were exposed to 5 min anoxia followed by 10 min reoxygenation. Chemical anoxia was introduced by Krebs-Henseleit(K-H) solution containing 10^-3 mol/L sodium dithionite. The spectrofluorometric method was used to verify intracellular calcium transient with fura-2/AM as calcium fluorescence probe. RESULTS: It was shown that during anoxia, the amplitude of Ca²⁺ transient was decreased, diastolic [Ca²⁺]_i, time to peak and time to relaxation of Ca²⁺ transient were increased. All the parameters were got back but did not returned to the pre-anoxia level during reoxygenation. IL-2 at 2×10⁵ U/L administrated during anoxia aggravated the effect of rexoxygenation on [Ca²⁺]_i transient. Pretreatment with a specific κ opioid antagonist, nor-BNI(10^-8 mol/L), abolished the effect induced by IL-2 during anoxia on the [Ca²⁺]_i transients, whereas specific δ opioid antagonist, naltrindole(10^-6 mol/L), did not cancel the effect. CONCLUSION: It is concluded that administration of IL-2 during anoxia aggravated the effect of reoxygenation on the [Ca²⁺]_i transients of isolated ventricular myocytes, which was mediated by cardiac κ opioid receptor pathway.     

Effect of oxymatrine on sodium channel in isolated ventricular myocytes in guinea pig

AIM: To observe the effect of oxymatrine on sodium channel in isolated cardiomyocytes of guinea pig. METHODS: Single ventricular myocytes of guinea pigs were obtained by enzymatic dissociation. The whole-cell patch clamp recording technique was used to record the change of sodium current by different dosage of oxymatrine from 1 to 1 000 μmol·L-1. RESULTS: Oxymatrine decreased sodium current in a dose-dependent manner. Oxymatrine (100 μmol·L-1) decreased the current density by 40% (P<0.01). The current-voltage curve was moved up and active potential, peak potential and reverse potential had no change. The inactivation curve was moved to more negative potential and the recovery curve was delayed. The activation curve had no significant change. CONCLUSION: Oxymatrine decreases the sodium channel current in a dose-dependent and voltage-dependent manner.     

p38 MAPK-Pim-3 signal pathway may be involved in cardiomyocyte anoxia preconditioning against anoxia/reoxygenation injury

AIM: To investigate the role of mitogen-activated protein kinases (MAPKs) pathways and the molecular mechanism by which the proto-oncogene Pim-3 protects cardiomyocyte against anoxia/reoxygenation (A/R) injury. METHODS: The primarily cultured neonatal rat ventricular cardiomyocytes were randomly divided into 4 groups: control group; A/R group; APC+A/R group; SB203850, U0126 or SP600125+APC+A/R group. The cells were pre-incubated with U0126 (ERK1/2 inhibitor), SP600125 (SAPK/JNK inhibitor), or SB203850 (p38 MAPK inhibitor) at concentration of 10 μmol/L for 30 min before the APC. The activities of p38 MAPK, JNK and ERK1/2 were detected by Western blotting. The viability of cardiomyocytes was assayed by MTT and the apoptosis of cardiomyocyte was detected by TUNEL. RESULTS: U0126, SB203850, and SP600125 abolished the increased expression of ERK1/2, p38-MAPK, and JNK proteins induced by APC+A/R or A/R, respectively. The expression level of Pim-3 protein significantly decreased when the p38 MAPK signal pathway was inhibited. Meanwhile, the activity of LDH and the apoptosis index increased, and the viability of cardiomyocytes decreased. CONCLUSION: Pim-3 expression through a p38 MAPK signaling pathway may protect cardiomyocytes from A/R injury.     

Effects of salvia miltiorrhizae on L-Ca current in ventricular myocyte of guinea pig during normoxia,acute hypoxia and reoxygenation

AIM: To observe the changes of Ca-L current (ICa-L) and to investigate the mechanism of salvia miltiorrhizae (SM) for eliminating Ca2+ overloaded in cells during acute hypoxia/reoxygenation. METHODS: The whole cell patch clamp technique was applied to study the changes of ICa-L. Different concentrations (32, 320, 3 200 mg/L) of SM were added to the ventricular myocytes isolated from guinea pigs by enzyme digestion. RESULTS: SM (32, 320, 3 200 mg/L) decreased the amplitude of ICa-L in a concentration-dependent manner regardless of these cells were under normoxia, hypoxia or reoxygenation. Furthermore, SM at low concentration (32 mg/L) was more effective to hypoxia or reoxygenation-treated cells than that to the cells under normoxia condition. CONCLUSION: These results indicate that SM effectively decreases the abnormal raised amplitude of ICa-L in ventricular myocytes under hypoxia or reoxygenation conditions, preventing Ca2+ overloaded in the cells.     

Effect of hypercholesterolemia on the ionic currents in cardiac ventricular myocytes of rats

AIM: To determine whether chronic hypercholesterolemia affects ionic currents on cardiac ventricular myocytes of rats. METHODS: Whole-cell patch-clamp technique was used to record the ionic currents in single cardiac myocytes isolated from normal cholesterolemia and hypercholesterolemia rats. RESULTS: In the hypercholesterol group (group Ⅱ), serum total-cholesterol level was significantly higher than that of normal group (group Ⅰ) ［(3.10±0.62)mmol·L-1 vs (1.18±0.37)mmol·L-1, P<0.01, n=20］. The serum triglyceride content of group II was remarkably higher than that of group Ⅰ ［(1.51±0.30)mmol·L-1 vs (0.43±0.15)mmol·L-1, P<0.01, n=20］. In ventricular myocytes of rats, 50% repolarization of action potential duration (APD50) prolonged from (70.86±8.12)ms (group Ⅰ) to (116.16±6.90)ms (group Ⅱ) (n=10 in each group, P<0.01); APD90 prolonged from (95.10±7.27)ms (group Ⅰ) to (144.04±7.39)ms (group Ⅱ) (n=10 in each group, P<0.01); at the test potential of -120 mV, Ik1 increased from (-16.98±4.54) pA/pF(group Ⅰ) to (-19.92±4.08) pA/pF (group Ⅱ) (n=12 in each group, P<0.05); at the test potential of 0 mV, ICa-L decreased from (-8.56±1.29) pA/pF (group Ⅰ) to (-5.24±0.90) pA/pF (group Ⅱ) (n=10 in each group, P<0.01); at the test potential of +60 mV, Ito decreased from (13.20±1.97) pA/pF (group Ⅰ) to (10.30±1.97) pA/pF (group Ⅱ) (n=8 in each group, P<0.05). CONCLUSION: Hypercholesterolemia affects the ionic currents on cardiomyocytes of rats greatly, which may be the ionic mechanism of cardiac toxicity induced by hypercholesterolemia.     

Interleukin-2 pretreatment improves the contractility of isolated rat ventricular myocytes during hypoxia and reoxygenation

AIM: To investigate the effect and possible mechanisms of interleukin-2 (IL-2) on the cell contractility in cardiomyocytes during hypoxia and reoxygenation.METHODS: Glucose-free Krebs-Henseleit (K-H) solution, gassed with 95% N2 and 5% CO2 for hypoxia, were used. The cell contractility were determined after 20 min of hypoxia and 30 min of reoxygenation by the video tracking system. The parameters of cell contractility included peak velocity of cell shortening (+dL/dtmax), peak velocity of cell relengthening (-dL/dtmax), contraction amplitude (dL) and end-diastolic cell length.RESULTS: It was shown that during hypoxia, the cell contraction was depressed. All the parameters were unable to return to the pre-hypoxia level during reoxygenation. Pretreatment with IL-2 at 2×103 U/L attenuated the inhibitory effect of hypoxia/reoxygenation on contractility in single ventricular myocytes. The effect of IL-2 was reduced in the presence of 10-8 mol/L nor-binaltorphimine (nor-BNI), a selective κ-opioid receptor antagonist. On blockade of protein kinase C with 3×10-6 mol/L chelerythrine, the effect of IL-2 was significantly attenuated. The effect of IL-2 was also blocked by 10-4 mol/L 5-hydroxydecanoate (5-HD), a mitochondrial ATP-sensitive potassium (KATP) channel blocker. CONCLUSIONS: The results of the present study provide evidence that pretreatment with IL-2 at 2×103 U/L attenuates the effect of hypoxia/reoxygenation on cell contraction in the isolated ventricular myocytes. The κ-opioid receptor mediates the effect of IL-2, in which activation of PKC and opening of mitochondrial ATP-sensitive potassium (mito KATP) channel are involved.     

Effect of platelet activating factor on the action potential and potassium currents in guinea-pig ventricular myocytes

AIM: To investigate the effects of platelet activating factor (PAF) on the action potential and potassium currents in guinea-pig ventricular myocytes. METHODS: By using whole-cell patch clamp technique, the effects of PAF on APD₉₀, I_K1 and I_K were investigated in enzymatically dispersed single guinea-pig ventricular myocytes. RESULTS: With 5 mmol/L ATP in the pipette electrode, 1 μmol/L PAF increased APD₉₀ from (225.8±23.3) ms to (352.8±29.8) ms (n=5, P<0.05), decreased I_K1 and I_K tail currents from (-6.1±1.3) nA to (-5.6±1.1) nA (n=5, P<0.05) at -120 mV and from (173.5±16.7) pA to (152.1±11.5) pA (P<0.05, n=4) at +30 mV, respectively. In contract, without ATP in the pipette electrode, 1 μmol/L PAF shortened APD₉₀ from (153.0±24.6) ms to (88.2±19.4) ms (n=5, P<0.01). Incubation of myocytes with 1 μmol/L glibenclamide, a blocker of I_KATP restored prolongation of APD induced by PAF. CONCLUSION: In guinea-pig ventricular myocytes, with 5 mmol/L ATP in the pipette, PAF prolonged APD partly due to the inhibition of I_K and I_K1, while with 0 mmol/L ATP in the pipette, PAF induced an activation of I_KATP, hence a decrease in APD was observed. Therefore, PAF might amplify the heterogeneity between ischemia and normal cardiac myocytes during ischemic reperfusion, which might play a vital role in the pathogenesis of the arrhythmias induced by ischemia/reperfusion.     

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.     

Effect of taurine on action potentials and ATP-sensitive posstiaum channel activity during hypoxia in ventricular muscle of guinea pig

AIM: To study the effects of taurine on ATP sensitive potassium channel (IK-ATP) during hypoxia in single ventricular myocyte. METHODS: The model of myocardial hypoxia was induced by unmixed and saturated nitrogen. IK-ATP activities were measured by whole-cell patch clamp recording. RESULTS: Activities of IK-ATP in the cell membrane of hypoxia ventricular myocyte significantly increased, compared to that in the normal. Extracellular injection of taurine (5，10，20 mmol/L) inhibited the increase in the IK-ATP activity in the hypoxia myocardium in a concentration-depend manner. Injection of taurine also recovered shorten APD during hypoxia. CONCLUSIONS: Taurine produces its cardioprotective effect by inhibiting the activity of IK-ATP in the hypoxia cardiomycytes of guinea pig. The results suggest that the depletion of taurine during myocardial hypoxia contributes to the early activation of the KATP channel.     

Effects of alcohol on L-type calcium channel of atrial myocytes in guinea pig

AIM: To observe the effect of acute alcohol intervention on atrial myocytic L-type calcium ion channels (ICa.L) in guinea pig by whole-cell patch clamp technique. METHODS: Guinea pig atrial myocytes were obtained by enzymolysis. Whole-cell patch clamp was used to gain the I-V curve, availability curve and activity curve of ICa.L with or without different concentrations of alcohol. RESULTS: The I-V curves of ICa.L were changed by alcohol in a concentration-dependent manner. Inhibition occurred when the alcohol concentration was less than 45 mmol/L, while enhancement was observed with a concentration more than 50 mmol/L. A sudden alternation between the two effects was observed with the alternation between the two concentrations. Alcohol concentration higher to 80 mmol/L did not change the I-V curve. Alcohol did not change the activity curve or availability curve of ICa.L except the former at concentration of 80 mmol/L. CONCLUSION: Alcohol affects ICa.L in a non-voltage-dependent way and a concentration-dependent manner where different concentrations of alcohol make inverse effects. That may help to elucidate the relationship between atrial fibrillation and atrial flutter.     

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.     

Effect of captopril on prolongation of action potential duration and reduction Ik in ischemia pig ventricular myocytes

AIM: To evaluate the effect of captopril on action potential duration and outward delayed rectification potassium current (Ik). METHODS: Action potentials were recorded using a conventional glass microelectrode filled with 3 mol/L KCl solution. Membrane patch clamp whole cell recording technique was used to investigate the Ik current maximum in the holding potential -50 mV, lasting time 100 ms, command potential +40 mV. RESULTS: The action potential duration of 30%, 50% repolarization (APD30, APD50) and ERP were significantly prolonged, but APD90 wasn't prolonged significantly when captopril group compared with ischemic group. The amplitude of Ik increased significantly in ischemic group, but significantly decreased in captopril group and in captopril+ischemic group. The shapes of current-voltage relationship were unchanged among groups, but significantly upward in ischemic group and downward in captopril and captopril+ischemic group. CONCLUSION: Captopril exerts electrophysiologic action due to decreasing delay outward rectification potassium current and prolonging action potential duration of APD30, APD50 and ERP.     

Role of voltage-dependent anion channel in cardiomyocytes subjected to anoxia/reoxygenation

AIM: To investigate the role of voltage-dependent anion channel (VDAC) in H9c2 cardiomyocytes subjected to anoxia/reoxygenation (A/R).METHODS: The shRNAs targeting VDAC1 mRNA were inserted into pSUPER plasmid.H9c2 cells were randomly divided into 5 groups as follows: control group, A/R group, anoxia preconditioning (APC) group, pSUPER-VDAC1-A/R group and pSUPER-A/R group. The expression of VDAC1 was detected by Western blotting. Cellular injury was evaluated by measuring the cell viability and the release of lactate dehydrogenase (LDH) and creatine phosphokinase (CPK). The mitochondria membrane potential was determined by flow cytometry.RESULTS: VDAC1 expression was up-regulated in A/R group and was inhibited in APC group. Similarly, down-regulation of VDAC1 expression by shRNA protected H9c2 cells from A/R injury. Moreover, we found that, with silencing VDAC1 expression, mitochondrial membrane potential was well preserved in H9c2 cells subjected to A/R.CONCLUSION: Down-regulation of VDAC1 protects H9c2 cells against A/R injury and its possible mechanism appears to be related to the regulation of mitochondial permeability transition pore opening.     

Effect of polyamines on intracellular calcium of the rat cardiomyocytes with anoxia and reoxygenation

AIM: To study the effect of extrogenous low concentration polyamine on cardiomyocyte calcium overload caused by anoxia and reoxygenation. METHODS: Enzymatically isolated rat ventricular myocytes were perfused with normal Tyrode solution for 8 min, then change to anoxia solution for 32 min, at last back to normal Tyrode solution perfusion for 8 min to establish the cardiomyocyte model of anoxia and reoxygenation. Spermine was added extracellularly to the bath before anoxia and spermine, spermidine or putrescine was added extracellularly after reoxygenation. Intracellular calcium fluorescence intensity (FI) was measured continuously by laser scan confocal microscope (LSCM). RESULTS: In the unstimulated state, exogenous spermine (1 mol/L) did not change resting ［Ca2+］i in the rat cardiomyocytes. Adding spermine before anoxia antagonized the ［Ca2+］i elevating caused by anoxia/reoxygenation. Adding spermine after reoxygenation also lowed the enhanced ［Ca2+］i caused by reoxygenation. Considering the potency of two conditions, the former was more efficacious than the later. Spermidine and putrescine also lowed the enhanced ［Ca2+］i caused by reoxygenation, but they were less efficacious than spermine. CONCLUSIONS: The results indicate that spermine given before anoxia or after reoxygenation, antagonized or lowed the cardiomyocytes calcium overload caused by anoxia/reoxygenation, but the later was weaker than the former. The order of potency of the polyamine lighten cardiomyocytes calcium overload caused by anoxia/reoxygenation was spermine>spermindine>putrescine.     

Alteration of Na+ currents in ventricular myocytes from 1-week infarcted rabbit heart

AIM:To study the current density and function of Na⁺ channel in cells from the epicardial border zone of the 1-week infarcted rabbit heart.METHODS:Rabbits were infarcted by ligation of the left anterior descending coronary artery.1 week later, INa was recorded using whole cel patch-clamp techniques in ventricular my ocytes from infarcted heart(IZs)and compared with the INa from noninfarcted heart(NZs).RESULTS:Peak I_Na current density(at -30 mV) was significantly reduced in IZs(22.48±4.62 PA/PF, n=14) compared with NZs(45.50±5.33 PA/PF, n=12), P＜0.01;V_0.5 of the availability curve(I/Imax curve)was shifted significantly in the hyperpolarizing direction in IZs(-89.1±5.6 mV, n=6) compared with NZs (-76.2±5.3 mV, n=5), P＜0.05.Recovery of I_Na from inactivation was significantly slower in IZs.CONCLUSIONS:I_Nais reduced, and its kinetics are altered in IZs.These changes may underlie the altered excitability and postrepolarization refractoriness of ventricular fibers of the IZs, thus contributing to reentrant arrhymias in the infarcted heart.     

Effect of AMPKα2 gene silencing by shRNA recombinant plasmid on chloride-mediated anoxia/reoxygenation injury in rat cardiomyocytes

AIM: To explore the role of AMP-activated protein kinase α2 subunit (AMPKα2) gene in chloride-mediated anoxia/reoxygenation (A/R) injury by transfection of short-hairpin RNA (shRNA) expression vector targeting to AMPKα2 gene into H9c2 cardiomyocytes. METHODS: Recombinant shRNA expression vector pSuper-AMPKα2 targeting to AMPKα2 gene was constructed and transfected into H9c2 cardiomyocytes. The protein expression of AMPKα2 was determined by Western blotting. The cells were divided into 5 groups: control group, A/R group, Cl^--free A/R group, pSuper+Cl^--free A/R group and pSuper-AMPKα2 shRNA+Cl^--free A/R group. After treatment, the cell viability was detected by MTT assay. LDH activity was analyzed with an automatic biochemical analyzer. The apoptotic rate and the level of intracellular ROS was measured by flow cytometry. The activity of SOD and GSH-Px was analyzed by a colorimetric method. RESULTS: The result of sequencing proved that the recombinant plasmid pSuper-AMPKα2 shRNA was correctly constructed. The protein level of AMPKα2 significantly decreased after the plasmid was transfected into the cardiomyocytes. Compared with A/R group, the cell viability and the activity of SOD and GSH-Px were significantly increased, while the activity of LDH, apoptotic rate and ROS production were significantly decreased in Cl^--free A/R group. The protective effect of Cl^--free solution on the A/R-induced injury of cardiomyocytes was abolished, and the ROS production was increased and the activity of SOD and GSH-Px was decreased after the cells were transfected with pSuper-AMPKα2 shRNA. CONCLUSION: Recombinant plasmid pSuper-AMPKα2 shRNA is successfully constructed, and silencing of AMPKα2 gene abolishes the protective effect of Cl^--free solution on A/R injury.     

Role of glycine receptor in protection of glycine against anoxia/reoxygenation injury in cardiomyocytes

AIM:To investigate whether glycine receptor is involved in the protection of glycine against anoxia/reoxygenation injury in cardiomyocytes by detecting oxygen free radical metabolism, apoptosis and intracellular calcium overload. METHODS:The neonatal rat cardiomyocytes were cultured and exposed to anoxia and reoxygenation (A/R) in the presence of glycine receptor antagonist, glycine or in free chloride buffer. The superoxide dismutase (SOD) activity, the contents of malondialdehyde (MDA) and nitric oxide (NO), the intracellular free calcium concentration and the apoptotic rate in the cardiomyocytes were determined. RESULTS:SOD activity and NO content in cardiomyocytes were lower, but MDA content, intracellular free calcium concentration and apoptotic rate in cardiomyocytes were higher in A/R group than those in control. Pretreatment with glycine inhibited the above changes caused by A/R, which was reversed by strychnine treatment and in the free chloride medium. CONCLUSIONS:Glycine inhibits free radical production, attenuates calcium overload, decreases apoptotic rate and increases SOD activity and NO release in cardiomyocytes exposed to A/R. These findings suggest that glycine exerts a protective effect against A/R injury via glycine receptor and glycine protects the neonatal rat cardiomycytes from A/R-induced injury in a chloride-dependent manner.     

Effect of garlicin on sodium channel current in rat ventricular myocytes

AIM: To observe the effect of garlicin on sodium channel(I_Na) in isolated ventricular myocytes of rats. METHODS: The ventricular myocytes of rats were obtained by enzymatic dissociation and treated with different concentrations of garlicin. The change of I_Na was recorded by the technique of whole-cell patch clamp recording.RESULTS: Garlicin decreased the I_Na of isolated ventricular myocytes in a dose- and voltage-dependent manner. The current density was elevated to peak under the condition that the membrane voltage was -40 mV before treated with garlicin. The current density was decreased by 48% from peak after treated with 500 μmol/L garlicin . No significant change of the active curve with the use of garlicin was observed. The median inactive voltages of the inactive curves before and after garlicin treatment were (-88.61±9.60) mV and (-103.03±8.90) mV (P<0.01), respectively, and the slopes were -7.85±0.88 and -7.55±2.75 (P>0.05), respectively, indicating that garlicin made a shift to the negative direction. CONCLUSION: Garlicin treatment induced the current density-voltage curve of I_Na to shift up and significantly decreased the current density. The inactive curve of sodium channel moved to the left, while the current density was not decreased after treated with garlicin. Garlicin blocks sodium channel in its inactive state in a dose- and voltage-dependent manner.