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Objective: To investigate whether inflammatory factor tumor necrosis factor-α (TNF-α) is involved in the electrical remodeling of cardiomyocytes by regulating ultra-rapid delayed rectifier K(+) current (I(kur)) and the role of Src kinase. Methods: H9c2 cells, embryonic cardiomyocytes of rat, were cultured in Dulbecco's modified Eagle's medium (DMEM) and atrium-derived HL-1 cells were cultured in Claycomb medium. Both H9c2 and HL-1 cells were cultured at 37 ℃ with 5% CO(2). Cells cultured in normal conditions without additional treatment served as control group. Experimental groups were treated with different concentration of TNF-α (25 or 50 or 100 ng/ml) for 24 hours. To study whether Src specific inhibitor PP1 could abrogate the effect of TNF-α, cells were pre-treated with 10 μmol/L PP1 for 1 hour, followed by TNF-α (100 ng/ml) for 24 hours. Western blot and the whole cell patch clamp technique were used to detect the protein expression of Kv1.5 and Src and I(kur) in each group. Results: (1) In H9c2 cells, high concentration of TNF-α treatment (100 ng/ml) significantly reduced the Kv1.5 protein expression compared with control group and TNF-α 25 ng/ml group (both P<0.05). Compared with control group, the expression of p-Src protein was higher in 25 ng/ml, 50 ng/ml, 100 ng/ml TNF-α group (all P<0.05), but there was no statistical difference in the expression of Src protein among groups (P>0.05). In addition, the current density of I(kur) was decreased in 50 ng/ml, 100 ng/ml TNF-α group (both P<0.05). Furthermore, the expression of Kv1.5 protein and the current density of I(kur) were increased in PP1+TNF-α group compared with TNF-α 100 ng/ml group (both P<0.05). There was no statistical difference in the expression of Kv1.5 protein and the current density of I(kur) between the control group and PP1+TNF-α group (both P>0.05). (2) In atrium-derived HL-1 cells, the expression of Kv1.5 protein was reduced in 100 ng/ml TNF-α group compared with control group and TNF-α 25 ng/ml group (both P<0.01). In addition, the expression of p-Src protein was increased in TNF-α 100 ng/ml group compared with control group (P<0.05), but there was no statistical difference in the protein expression of Src among groups (P>0.05). The expression of Kv1.5 protein was increased in PP1+TNF-α group compared with TNF-α 100 ng/ml group (P<0.05). Conclusion: TNF-α is involved in the pathogenesis of atrial fibrillation, probably via decreasing I(kur) current density in atrium-derived myocytes through the activation of Src kinase.
Subject(s)
Animals , Rats , Down-Regulation , Heart Atria , Myocytes, Cardiac , Tumor Necrosis Factor-alpha , src-Family KinasesABSTRACT
Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca²⁺ homeostasis. The store-operated calcium (SOC) channel is the primary Ca²⁺ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca²⁺ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca²⁺ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.
Subject(s)
Blotting, Western , Calcium , Endoplasmic Reticulum , Endoplasmic Reticulum Stress , Endothelial Cells , Homeostasis , Human Umbilical Vein Endothelial Cells , Tunicamycin , Unfolded Protein ResponseABSTRACT
Aim To study whether there was arterial heterogeneity and association with L-type calcium channel (LCC) in different parts of arteries in re-sponse to certain vasoconstrictor. Methods The aor-ta, renal arteries and coronary arteries were dissected from rats. Arterial ring contractions induced by pheny-lephrine (Phe), 5-hydroxyl tryptamine (5-HT) or U46619 in concentration-dependent manner were meas-ured using the Multi Myograph system and the response to nifedipne was observed. Results (1) Phe had no obvious effect on the tension of coronary artery,but in-duced concentration-dependent vasoconstriction in aor-ta and renal artery,and pEC50of aorta was significantly higher than that of renal artery (P<0.05). The inhi-bition rate of nifedipine on the aortic contractile re-sponses was significantly higher than that of renal arter-y (P<0.05). (2) The contraction induced by 5-HT on aorta was not obvious, but was significant on renal artery and coronary artery. The inhibitory rate of nife-dipine on coronary artery vasoconstriction was signifi-cantly higher than that of renal artery (P <0.05). (3) U46619 could induce aorta,renal artery and coro-nary artery concentration- dependent contraction, but the Emaxof them were both higher than that of renal ar-tery (P<0.05). And the pEC50of aorta was the lar-gest (P<0.05). Nifedipine significantly inhibited the contraction of aorta, renal artery and coronary artery induced by U46619 with the greatest inhibitory rate on the coronary artery vasoconstriction and minimal inhibi-tion on aortic vasoconstriction. Conclusions The re-sponse to certain vasoconstrictor is different among aor-ta, renal artery and coronary artery in rats, and the contraction mediated by L-type calcium channel is also different.
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AIM:To investigate the possible mechanism of coronary artery contraction induced by 5-hydroxytryptamine(5-HT).METHODS:Isolated coronary artery rings were obtained from male Wistar rats,and the vas-cular tension meter was used to determine the tension of the coronary artery rings.The effects of inhibitors of different sig-naling pathway on vascular contraction tension induced by 5-HT were observed.RESULTS:Firstly,we found that 5-HT2A receptor antagonist sarpogrelate(1 μmol/L)completely eliminated the coronary artery contraction induced by 5-HT.Phos-pholipase Cβ(PLCβ)inhibitor U73122(10 μmol/L and 50 μmol/L), Rho-related protein kinase inhibitor Y-27632(3 μmol/L and 10 μmol/L)and protein kinase C δ subunit(PKCδ)inhibitor rottlerin(3 μmol/L and 10 μmol/L)signifi-cantly inhibited the contraction of coronary artery ring caused by 5-HT(P<0.05).In addition, compared with the un-treated group,vascular contraction tension induced by 5-HT was also decreased significantly by L-type calcium channel (Cav1.2)blocker nifedipine(1 μmol/L), store-operated Ca2+entry(SOCE)inhibitor SKF96365(10 μmol/L and 30 μmol/L)and 2-aminoethoxydiphenyl borate(2-APB,50 μmol/L and 100 μmol/L)(P<0.05).At the same time,5-HT also induced vasoconstriction after treated with nifedipine(1 μmol/L)Kerbs-Henseleit(K-H)liquid without calcium (P<0.05).CONCLUSION:5-HT activates 5-HT2Areceptor induced coronary artery contraction,possibly related to the PKC/Rho kinase signaling pathway and calcium regulation.
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AIM:To investigate the changes of cardiac structure and function in rats with type 2 diabetic melli-tus(T2DM),and to explore the mechanisms underlying diabetic cardiomyopathy.METHODS:The cardiac structure and function were measured by echocardiography in Zucker diabetic fatty(ZDF)rats and their control Zucker lean(ZL)rats. The size of the cardiomyocytes was determined by wheat germ agglutinin staining.The protein expression of atrial natriuretic peptide(ANP),β-myosin heavy chain(β-MHC), receptor for advanced glycation end products(RAGE), L-type cal-cium channel α1C subunit(CaV1.2)and Orai1 was assessed by Western blot.RESULTS:Compared with the ZL control rats,the thickness of left ventricular wall,ejection fraction(EF),fractional shortening(FS)and the sizes of cardiomyo-cytes were significantly increased,and diastolic function was decreased in the ZDF rats(P<0.05).The protein expression of β-MHC, ANP, RAGE and Orai1 was increased, while the expression of Ca V1.2 was decreased in ZDF rats(P <0.05).CONCLUSION:T2DM rats show the prominent features including cardiomyocyte hypertrophy,ventricular hyper-trophy and compensatory enhancement of cardiac function, and the Ca2+handling and increase in RAGE expression may play important roles in the processes.
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AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress ( ERS) model in CASMCs of SD rats.METHODS:CASMCs were cultured by tissue explant method .The morphological characteristics were observed under optical micro-scope.The marker proteins of CASMCs , including α-SMA and SM-MHC, were identified by immunofluorescence tech-nique.The protein expression levels of BiP and CHOP , the marker molecules of ERS, were determined by Western blot . RESULTS:The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical hill and valleygrowth pattern of CASMCs at 9~10 d.The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed .The results of Western blot showed that the protein expression of BiP and CHOP in TG ( 1 and 2 μmol/L ) treatment groups was increased compared with control group .Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION:CASMCs can be successfully cultured by tissue explant method .ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h.
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AIM:To determine circular RNA (circRNA) profiles in the diabetic mouse myocardium , and to investigate the effect of circRNA_000203 on fibrotic phenotypes in cardiac fibroblasts .METHODS:Masson trichrome stai-ning was performed on the myocardium of the diabetic db /db mice and the non diabetic db/m control mice .circRNA ex-pression profile in the diabetic myocardium was detected by circRNAs microarray .The expression of circRNA_000203 was determined by real time fluorescence quantitative PCR ( RT-qPCR ) .Recombinant circRNA_000203 adenovirus was pre-pared for enforced the expression of circRNA_000203 in mouse cardiac fibroblasts.The expression of Col1a2, Col3a1andα-SMA was determined in circRNA_000203-modified cardiac fibroblasts , respectively .RESULTS:Masson trichrome stai-ning showed that fibrosis was increased in the diabetic mouse myocardium .The results of circRNA array detection revealed that circRNAs were dysregulated in the diabetic myocardium .circRNA_000203 was up-regulated in the diabetic myocardi-um.Significant over-expression of circRNA_000203 was achieved in the cardiac fibroblasts after infection with the recombi-nant circRNA_000203 adenovirus.The mRNA and protein expression of Col1a2, Col3a1 and α-SMA was significantly in-creased in the cardiac fibroblasts with over-expression of circRNA_000203.CONCLUSION:circRNA_000203 is up-regu-lated in the diabetic mouse myocardium .It has pro-fibrotic effect on the cardiac fibroblasts .
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AIM:Increasing evidence indicates that inflammation contributes to the initiation and perpetuation of atrial fibrillation ( AF) .Al-though tumor necrosis factor ( TNF)-αlevels are increased in patients with AF , the role of TNF-αin the pathogenesis of AF remains unclear.Recent research has revealed that T-type Ca2+currents ( ICa,T ) play an important role in the pathogenesis of AF .METH-ODS:In this study , we used the whole-cell voltage-clamp technique and biochemical assays to explore the role of TNF-αin the regula-tion of ICa,T in atrial myocytes.RESULTS:We found that compared with sinus rhythm (SR) controls, T-type calcium channel (TCC) subunit mRNA levels were decreased , while TNF-αexpression levels were increased , in human atrial tissue from patients with AF .In murine atrial myocyte HL-1 cells, after cultured for 24 h, 12.5, 25 and 50 μg/L TNF-αsignificantly reduced the protein expression levels of the TCC α1G subunit in a concentration-dependent manner .The peak current was reduced by the application of 12.5 or 25μg/L TNF-αin a concentration-dependent manner [from ( -15.08 ±1.11) pA/pF in controls to ( -11.89 ±0.83) pA/pF and (-8.54 ±1.55) pA/pF in 12.5 and 25 μg/L TNF-αgroups, respectively].TNF-αapplication also inhibited voltage-dependent inactivation of ICa,T shifted the inactivation curve to the left .CONCLUSION:These results suggest that TNF-αis involved in the path-ogenesis of AF, probably via decreasing ICa,T function in atrium-derived myocytes through impaired channel function and down -regula-tion of channel protein expression .This pathway thus represents a potential pathogenic mechanism in AF .
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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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<p><b>OBJECTIVE</b>To investigate the association of desmoplakin with the distribution and function of Nav1.5 by RNA silencing technology in HL-1 cells.</p><p><b>METHODS</b>HL-1 cells with desmoplakin expression suppression by RNA silencing were examined for desmoplakin and Nav1.5 protein expressions by Western blotting, and the distribution and co-location of desmoplakin and Nav1.5 protein were detected by immunofluorescence staining. Patch-clamp recording was applied to analyze the changes in whole-cell sodium current after desmoplakin silencing.</p><p><b>RESULTS</b>Compared with the untreated group and negative control group, the cells with desmoplakin silencing showed obviously reduced expressions of desmoplakin and Nav1.5 proteins. Co-localization of desmoplakin and Nav1.5 was detected at cell-cell contact in untreated and control conditions, and desmoplakin expression silencing induced a drastic redistribution of Nav1.5 with decreased peak current density (156.3∓6.2 vs 41.8∓3.1, n=6, P<0.05), a shift in voltage dependence of steady-state inactivation (-42 mV vs -61 mV, n=5, P<0.05), and prolonged time of recovery from inactivation.</p><p><b>CONCLUSION</b>Desmoplakin silencing caused redistribution of Nav1.5 protein and also changes in its electrophysiological properties in HL-1 cells.</p>
Subject(s)
Animals , Mice , Cell Line , Desmoplakins , Genetics , Metabolism , Gene Silencing , Mutation , Myocytes, Cardiac , Metabolism , MetabolismABSTRACT
AIM: To observe the effects of high hydrostatic pressure on asymmetric NG, NG-dimethyl-L-arginine (ADMA) metabolism of human vascular endothelial cells (HUVECs), and the role of renin-angiotensin system (RAS). METHODS: Cultured HUVECs of 3-6th passage were exposed to atmosphere (0 mmHg, APC), 120 mmHg (MPC), 180 mmHg (HPC). There were three groups in each pressure condition, one as control, the other two were interfered with captopril (Cap, 10 ?mol/L or 100 ?mol/L) or irbesartan (Irb, 10 ?mol/L or 100 ?mol/L) respectively. Cell proliferation was quantified by determining hexosaminidase activity at 12 h. Concentration of ADMA in conditioned medium was measured by high performance liquid chromatography (HPLC) at 12 h. RESULTS: Compared with APC group, ADMA concentration increased prominently in MPC and HPC (4.69?0.37 and 4.48?0.39 vs 0.75?0.05,P
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AIM: To observe the effects of oxygen free radical (OFR) and captopril on the level of asymmetric NG, NG-dimethyl-L-arginine (ADMA) in human vascular endothelial cells (HUVECs).METHODS: HUVECs of 3-6 th passage, cultured with modified Jaffes' method, were used in the experiment and divided into three groups: (1)Cells cultured with equivalence of DMEM medium as control; (2)OFR intervention groups, OFR at concentrations of 0 01 mmol/L, or 0 1 mmol/L, respectively, were added to the cell culture; (3)Drug intervention groups: the cell culture was treated with 0 1 mmol/L of OFR combined with 50 mg/L or 100 mg/L of captopril, respectively. Concentrations of ADMA, L-arginine, nitric oxide(NO), endothelin(ET) and the activity of angiotensin-converting enzyme(ACE) in conditioned medium were measured after 24 h exposure. RESULTS: Concentrations of ADMA, ET and the activity of ACE were increased, while the amount of NO decreased in OFR intervention groups compared with control group. After treatment with captopril, ADMA, ET concentrations and the activity of ACE were decreased, while the amount of NO increased, but the level of L-arginine had no obvious change. CONCLUSIONS: OFR induces endothelial dysfunction through increasing ADMA concentration, while captopril relieves endothelial dysfunction induced by ox-LDL through decreasing ADMA concentration.
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AIM: To investigate whether or not short-term high pressur e proliferates human umbilical vein endothelial cells (HUVECs), and the role of pp60c-src in cell proliferation. METHODS: Cultured HUVECs of 3-6th passa ge were exposed to atmosphere 0 mmHg (AP), 120 mmHg (MP), 180 mmHg (HP) and interfered with cap top ril(Cap, 10 ?mol/L)or irbesartan(Irb, 10 ?mol/L) Cell proliferation was q uantified by measuring hexosaminidase activity. The content of pp 60 c-src in cells was investigated by Western blotting. RESULTS:① Hexos aminidase activities which reflected cell number increased significantly at 4 h in MP and HP(0 145?0 018 and 0 144?0 019 vs 0 118?0 003,P