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AIM:To observe the effects of granulocyte colony-stimulating factor (G-CSF) on calcium, sodium and potassium ion channel currents of the ischemic atrial myocytes in guinea pig by whole-cell patch clamp technique.METHODS:The guinea pig atrial myocytes were obtained by enzymolysis.Under ischemia and hypoxia condition, whole-cell patch clamp was used to observe the effects of G-CSF at various concentrations on the changes of the I-V curve, activation curve and availability of L-type calcium channel current (ICa,L) and voltage-dependent sodium channel current (INa), as well as I-V curve of delayed rectifier potassium channel current (IK).RESULTS:Under ischemic condition, the I-V curves of ICa,L were changed by acute G-CSF intervention in a dose-dependent fashion.Except for G-CSF at dose of 300 μg/kg, the other concentrations of G-CSF did not change the activation curve and availability of ICa,L, indicating that the effects of G-CSF on ICa,L were in a voltage-independent fashion.The I-V curves of ICa,L under ischemic condition were gradually approaching the normal levels by the higher dose of G-CSF, while the effect of 300 μg/kg G-CSF on ICa,L was similar to 100 μg/kg G-CSF.Acute G-CSF intervention at different doses did not change I-V curve, activation curve, and availability or steady-state availability of INa.As a part of IK, the rapid activating component (IKr) was improved by 100 μg/kg and 300 μg/kg G-CSF intervention with the similar effects, while the slowly activating component (IKs) was not changed by G-CSF.CONCLUSION:G-CSF affects ion channel electrophysiological properties of ischemic atrial myocytes in a voltage-independent but concentration-dependent manner, thus reducing the incidence of atrial arrhythmia.
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Objective To study the effect of allicin ( All) on potassium current in single atrial myocytes in rats. Methods Isolated rat atrial myocytes were isolated by perfusion and administered by extracellular perfusion method.Whole cell patch clamp technique was used to record transient outward potassium current ( Ito ) in atrial myocyte. Results In presence of 30 μmol·L-1 of All,the peak current of Ito was significantly reduced from (20.5±2.2) pA/pF to (11.3±2.1) pA/pF at+50 mV of test potential (P<0.01,n=12).This effect of All showed voltage- and concentration-dependence with IC50 of (19.0±2.5)μmol·L-1 .The steady-state activation curve of Ito was shifted to more positive potential and recovery time from inactivation was prolonged.In addition, they fail to find any effect of All on the steady-state inactivation and closed-state inactivation of Ito. Conclusion All can inhibit Ito by slowing down the process of activation and recovery from inactivation of channel.
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Objective To study the effect of allicin ( All) on potassium current in single atrial myocytes in rats. Methods Isolated rat atrial myocytes were isolated by perfusion and administered by extracellular perfusion method.Whole cell patch clamp technique was used to record transient outward potassium current ( Ito ) in atrial myocyte. Results In presence of 30 μmol·L-1 of All,the peak current of Ito was significantly reduced from (20.5±2.2) pA/pF to (11.3±2.1) pA/pF at+50 mV of test potential (P<0.01,n=12).This effect of All showed voltage- and concentration-dependence with IC50 of (19.0±2.5)μmol·L-1 .The steady-state activation curve of Ito was shifted to more positive potential and recovery time from inactivation was prolonged.In addition, they fail to find any effect of All on the steady-state inactivation and closed-state inactivation of Ito. Conclusion All can inhibit Ito by slowing down the process of activation and recovery from inactivation of channel.
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AIM: To detect the effects of resveratrol (RSV) on the expression of microRNA-21 (miR-21) in primarily cultured neonatal rat atrial myocytes with electric remodeling induced by rapid electrical stimulation (RES).Furthermore, to find out the possible mechanism of miR-21 regulating electrical remodeling.METHODS: The neonatal rat atrial myocytes were isolated by double-enzyme (trypsin and collagenase I) digestion and differential adhesion method.The atrial fibrillation (AF) model was induced by RES.Atrial myocytes were randomly divided into 4 groups: control group, RSV group, RES group, and RSV+RES group.To further detect whether RSV regulated electric remodeling by miR-21, except the 4 groups, we add miR-21 over-expression group and miR-21 inhibitor group: RES+negative control (NC) group, RES+miR-21 mimics group, RES+miR-21 mimics+RSV group, RES+miR-21 inhibitor group, and RES+miR-21 inhibitor+RSV group.The optimal concentration and pretreatment time of resveratrol were determined by CCK-8 assay.The expression of miR-21 and the mRNA expression of L-type calcium channels CACNA1C and CACNB2 in atrial myocytes were detected by qPCR.The protein expression of L-type calcium channels Cav1.2 and Cavβ2 in the atrial myocytes was analyzed by Western blot.RESULTS: The expression of miR-21 in RES group was significantly increased compared with control group, while preconditioning with RSV decreased the expression of miR-21.Compared with RES+miR-21 mimics group, the expression of miR-21 in RES+miR-21 mimics+RSV group was significantly decreased.Meanwhile, the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ2 were increased (P<0.05).Compared with RES group, the expression of miR-21 in RES+miR-21 inhibitor group and RES+miR-21 inhibitor+RSV group was decreased, while the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ2 were increased.However, no difference of the expression of miR-21, the mRNA expression of CACNA1C and CACNB2, and the protein levels of Cav1.2 and Cavβ2 among RSV+RES, RES+miR-21 inhibitor and RES+miR-21 inhibitor+RSV groups was observed (P<0.05).CONCLUSION: In AF model induced by RES, RSV may reduce electric remodeling by inhibiting the expression of miR-21 and regulating the downstream target genes.
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AIM:To investigate whether the association of connexin 43 ( Cx43 ) and L-type calcium channel involved in the pathogenesis of atrial fibrillation ( AF) .METHODS:The biochemical assays and whole-cell patch-clamp technique were used to study the expression of Cx43 in human atrial tissue.The co-localization of Cx43 and L-type calcium channel, and the regulation of L-type calcium current in atrial myocytes were investigated.RESULTS:The expression of Cx43 at mRNA and protein levels was decreased in human atrial tissues of AF patients.In cultured atrium-derived myocytes ( HL-1 cells) , knockdown of Cx43 significantly inhibited the mRNA expression of L-type calcium channelα1c subunit, as well as L-type calcium current.Co-localization of Cx43 with L-type calcium channel α1c subunit in mouse atrial myocytes was observed.CONCLUSION:The decrease in Cx43 is involved in the pathogenesis of AF, probably through reducing the L-type calcium current in atrial myoctyes by co-localization with L-type calcium channel, thus representing the potential pathogenesis in atrial fibrillation.
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Objective To establish a stable method for acute isolation of atrial myocytes in diabetic rats,so as to provide materials for electrophysio?logical study of diabetic rats. Methods Streptozotocin(STZ)was used to establish type I diabetic model. Atrial myocytes were isolated with modi?fied perfusion buffer by Langendorff perfusion. The atrial myocytes were morphologically observed with optical microscope and identified by morphol?ogy and immunofluorescence staining. The action potential was recorded by patch clamp technique. Results STZ can establish a stable type I dia?betic model. The modified perfusion method can yield calcium tolerant and spindle shaped atrial myocytes. Immunofluorescence indicated that atrial myocytes were positive for Kv1.5 which was expressed in atrial myocytes. The atrial myocytes obtained by this method were able to generate action po?tentials. Conclusion The modified perfusion method is suitable for acute isolation of atrial myocytes in rats with diabetes mellitus,which may help to study the electrophysiology of diabetic heart.
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Objective To explore the condition and method of primary cultures of atrial myoeytes in sukling guinea pig .Methods The atrial myoeytes of sukling guinea pig with 1‐2 d old were taken and digested once by 0 .06% trypsin ,then repeatedly digested by 0 .08% type Ⅱ collagenase for four times .And the cells after former twice ,latter twice and former and latter four times of diges‐tion were adopted for conducting respective culture .The influence of three kinds of culture method on atrial myocytes production , survival rate ,pulsating rate and purification rate were observed .Results Three kinds of culture method all obtained the considera‐ble number of atrial myocytes and a higher survival rate ,pulsation rate and purification rate ,particularly the cell culture method af‐ter latter twice digestion .Conclusion Once digestion by 0 .06% trypsin and 0 .08% type Ⅱ collagenase for four times can isolate a great quantity and good condition of atrial myocytes .
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[ABSTRACT]AIM:Toinvestigatetheeffectsofmechanicalstretchontransientoutwardpotassiumcurrent(Ito), inward rectifier potassium current ( IK1 ) and action potential duration ( APD) of cultured neonatal rat atrial myocytes . METHODS:Neonatal rat atrial myocytes were isolated and cultured on silicone sheeting with or without stretch for 24 h. The silicone membrane area was increased by 12%in stretched group.The cells without stretch served as control .Ito, IK1 and APD were recorded by the technique of whole-cell patch clamp.RESULTS:Compared with control group, Ito density in stretched myocytes was significantly reduced [(1.6 ±0.4) pA/pF vs (12.1 ±2.9) pA/pF, P<0.01], whereas IK1 density was increased [(-10.8 ±0.8) pA/pF vs (-8.8 ±0.9) pA/pF, P<0.01].The APDs at 50%and 90%levels of repolarization ( APD50 and APD90 ) in the stretched cells were obviously decreased than those in non-stretched cells [(10.5 ±1.4) ms vs (15.5 ±2.4) ms, (30.0 ±2.8) ms vs (56.3 ±3.6) ms, P<0.01].CONCLUSION: Stretch stimulation leads to the reduction of Ito density, the increase in IK1 density and the shortness of APD in cultured rat atrial neonatal myocytes , which may contribute to atrial electrical remodeling induced by pressure overload .
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Mechanical stimuli to the cardiac myocytes initiate many biochemical and physiological events. Stretch-activated cation channels have been suggested to mediate these events. In this study, cell-attached and inside-out excised-patch clamp methods were used to identify stretch-activated cation channels in adult rat atrial myocytes. Channel openings were increased in cell-attached configuration when negative pressure was applied to the pipette, and also in inside-out excised patches by negative pressure. The channel was not permeable to Cl-, Na+ and Cs+, but selectively permeable to K+, and the degree of activation was dependent on the magnitude of negative pressure (full activation at ~ -50 mmHg). In symmetrical 140 mM KCl, the slope conductance was 51.2+/-3 pS between the potentials of -80 and 0 mV and 55+/-6 pS between 0 and +80 mV (n=5). Glibenclamide (100 microM) or ATP (2 mM) failed to block the channel openings, indicating that it is not ATP-sensitive K+ channel. Arachidonic acid (30 microM), which has been shown to activate a K+ channel cooperatively with membrane stretch, did not affect the channel activity. GdCl3 (100 microM) also did not alter the activity. These results demonstrate that the mechanical stretch in rat atrial myocytes activates a novel K+ -selective cation channel, which is not associated with other K+ channels such as ATP-sensitive and arachidonic acid-activated K+ channel.
Subject(s)
Adult , Animals , Humans , Rats , Adenosine Triphosphate , Arachidonic Acid , Glyburide , Membranes , Muscle Cells , Myocytes, CardiacABSTRACT
The atrial acetylcholine-activated K+ (KACh) channel is gated by the pertussis toxin-sensitive inhibitory G (GK) protein. Earlier studies revealed that ATP alone can activate the KACh channel via transphosphorylation mediated by nucleoside-diphosphate kinase (NDPK) in atrial cells of rabbit and guinea pig. This channel can be activated by various agonists and also modulated its function by phosphorylation. ATP-induced KACh channel activation (AIKA) was maintained in the presence of the NDPK inhibitor, suggesting the existence of a mechanism other than NDPK-mediated process. Here we hypothesized the phosphorylation process as another mechanism underlying AIKA and was undertaken to examine what kinase is involved in atrial cells isolated from the rat heart. Single application of 1 mM ATP gradually increased the activity of KACh channels and reached its maximum 40 ~ 50 sec later following adding ATP. AIKA was not completely reduced but maintained by half even in the presence of NDPK inhibitor. Neither ADP nor a non-hydrolyzable ATP analogue, AMP-PNP can cause AIKA, while a non-specific phosphatase, alkaline phosphatase blocked completely AIKA. PKC antagonists such as sphingosine or tamoxifen, completely blocked AIKA, whereas PKC catalytic domain increased AIKA. Taken together, it is suggested that the PKC-mediated phosphorylation is partly involved in AIKA.
Subject(s)
Animals , Rats , Adenosine Diphosphate , Adenosine Triphosphate , Adenylyl Imidodiphosphate , Alkaline Phosphatase , Catalytic Domain , Guinea Pigs , Heart , Nucleoside-Diphosphate Kinase , Phosphorylation , Phosphotransferases , Protein Kinase C , Protein Kinases , Sphingosine , Tamoxifen , Whooping CoughABSTRACT
Brain natriuretic peptide(BNP) is a recently discovered novel neuropeptide of 26 amino acid residues, isolated from porcine brain, that is of similar potency to atrial natriuretic factor(ANF) in natriuretic, diuretic, hypotensive and smooth muscle relaxant activities. we used a highly selective antisera against BNP raised in rabbit to observe its distribution and localization in some brain areas and some other peripheral tissues by utili zing high sensitive avidin-biotin-peroxidase complex technique. Positive brain natriuretic peptide immunoreactive (BNPir) fibers and cell bodies were observed in the lateral hypothalamic area, caudate-putamen, hippocampus, amygdala, and supraoptic and paraventricular nuclei. A lot of BNPir granules were also found in rat atria. They were of similar localization to that of ANF. Most of the specific granules were accumulated in cytoplasm at both nuclear poles of atrial myocytes. The BNP immunoreactivity is less intense than that of the ANF. Some of scanty, diffuse and fine BNPir granules could also be observed in ventricular myocytes.The coexistence of both BNP and ANF in the brain and heart indicates that BNP may function as a neuropeptide and circulating hormone, and suggests the possibility that the physiological effects such as diuretic natriuretic, hypotensive and smooth muscle relaxant activities so far thought to be mediated by ANF may be regulated through a dual mechanism involving both BNP and ANF. In addition, some BNPir positive cells were also present in the anterior and intermediate lobes of rat pituitary gland. The significance of BNP in hypophysis would be elucidated in the further studies.
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6 months; Control group (n=8) without AF. Atrial muscle samples of the left and right atrial appendage were cut off during heart valve replacement. Morphological changes were observed by light microscopy and electron microscopy. Results ① The incidence rates of Aschoff body were 37.5% (3/8), 46% (5/11) , and 50% (6/12) in Group Ⅰ, Group Ⅱ, and the control group. There was no difference in Aschoff body between the left and right atrial myocytes. ② There was no difference in myocytic degeneration, interstitial hyperplasia, and lymphocytes infiltration between the left and right atrial myocytes in each group. ③ Myolysis, accumulation of glycogen, and fibrosis were detected by electron microscopy in the 3 groups. Changes of myolysis and fibrosis were more obvious in patients with AF than patients without AF. Conclusion There are similar pathological changes in the left and right atria in rheumatic heart disease patients with or without AF. Fibrosis of atrial myocytes may be involved in the occurrence and maintenance of AF.