| Part Ⅰ:Indoxyl sulfate reduces Ito,f current and Ito,f current-related protein expression levels in vitroObjective:To investigate the effects of indoxyl sulfate(IS)on the fast transient outward potassium current(Ito,f)and action potential(AP).Methods:In this study,neonatal rat ventricular myocytes(NRVM)were successfully extracted and divided into control group,IS0.1μ M group,IS 1μ M group,IS 10 μM group and IS 100 μM group.Then the viability of NRVM was detected by trypan blue staining,and the number of troponin I positive cells was detected by immunofluorescence technique to identify the purity of cardiomyocytes.The activity of cardiomyocytes was detected by MTT assay.The mRNA and protein expression levels of Kv4.2,Kv4.3 and KChIP2 in NRVM were detected by qRT-PCR and Western blot,respectively.Then,in the whole cell voltage clamp mode,Ito,f currents of different groups were recorded.The activation and decay curves of Ito,f currents were fitted by Boltzmann and monoexponential equations respectively,and the constants of activation and decay were calculated.In the whole cell current clamp mode,AP trajectories were recorded and APD50,APD90 values were calculated.In addition,the relationship between the reduction of Ito,f current caused by IS and AP is further studied by using the computer virtual model of AP.Results:1.The spontaneous beat frequency of NRVM was 80-110 beats/min,the survival rate was 95.45%±0.35%,and the purity was 94.60±1.17%.The activity of NRVM was not affected by different concentration gradient of IS(P>0.05).2.With the increase of IS concentration,the mRNA and protein expression levels of Kv4.3,Kv4.3 and KChIP2 decreased.3.The Ito,f current density(15.54±0.23,12.33±0.19,8.31±0.41 and 3.27±0.24)of ISO.1 μM,IS1 μM,IS10μM and IS100 μM groups were significantly lower than that of the control group(19.09±0.51)when the command voltage was 60 mV(P<0.01).4.After fitting with Boltzmann equation and monoexponential equations,the values of current activation constant V0.5,k and decay constant τ decrease with the increase of IS concentration.5.The APD50 value of IS group(305.08±26.40)was significantly higher than that of the control group(151.03±15.98)(P<0.01).The APD90 value(521.40±32.49)of the IS group was also significantly higher than that of the control group(278.00±16.54)(P<0.01).The effect of IS on Ito,f current was simulated by computer virtual model of action potential,it can be seen that IS also prolongs APD.Conclusions:1.Indoxyl sulfate decreased the mRNA and protein expression levels of Kv4.2,Kv4.3 and KChIP2.2.Indoxyl sulfate reduced the Ito,f current in a dose-dependent manner.3.Indoxyl sulfate prolonged the action potential duration.Part Ⅱ:Indoxyl sulfate reduces Ito,f current through ROS/MAPK and NF-κB signaling pathwaysObjective:To explore the potential mechanism of indoxyl sulfate reducing the Ito,f current.Methods:After successfully separating NRVM,Control group,IS group,NAC group,DPI group,SB group,U0126 group,BAY group,IS+NNAC group,IS+DPI group,IS+SB group,IS+U0126 group and IS+BAY group were set respectively.After that,the reactive oxygen species(ROS)was determined based on flow cytometry,and the expression levels of Kv4.2,Kv4.3 and KChIP2 mRNA in each group were detected by qRT-PCR technology,the expression levels of Kv4.2,Kv4.3,KChIP2,NOX2,Phospho-p38 MAPK,p38 MAPK,Phospho-p44/42 MAPK(Erk1/2),p44/42 MAPK(Erkl/2),Phospho-NF-κB-p65 and NF-κB-p65 were detected by Western blot.A laser scanning confocal microscope was used to observe the translocation of NF-KB-p65 protein from the cytoplasm to the nucleus.Finally,the patch clamp technique was used to record the Ito,f current in each group,the activation and decay constants of current were analyzed,and AP trajectories were also recorded.Results:1.IS increased the fluorescence intensity of ROS in a dose-dependent manner,and the ROS scavenger NAC partially reversed the increase in ROS production caused by IS.2.The expression of NOX2 protein gradually increased with the increase of IS concentration.The NAD(P)H oxidase inhibitor DPI partially reversed the activation effect of IS on NOX2 protein.3.The relative expression levels of mRNA and protein of Kv4.2,Kv4.3 and KChIP2 in the IS+NAC group were significantly higher than those in the IS group(all P values<0.01).5.ROS scavenger NAC can partially reverse the decrease of Ito,f current induced by IS.When the command voltage was 60mV,the IS+NAC group(9.83±0.30)was significantly larger than the IS group(7.93±0.34)(P<0.05),6.The activation constant V0.5 value and k value of the IS+NAC group were significantly higher than that of the IS group(both P values<0.05).The τ value of the IS+NAC group was also significantly higher than that of the IS group(P<0.01).7.The APD50 value of the IS+NAC group(207.17±9.12)was significantly lower than that of the IS group(337.18±27.54)(P<0.01).Similarly,the APD90 value of the IS+NAC group(354.80±4.01)was also significantly lower than that of the IS group(548.62±22.71)(P<0.01).8.The protein expression levels of P-p38 MAPK,P-p44/42 MAPK,P-NF-κB,p38 MAPK,p44/42 MAPK and NF-κB were detected by Western blotting technology.It can be seen that IS can increase the phosphorylation level of p38 MAPK,p44/42 MAPK and NF-κB protein in NRVM in a dose-dependent manner.Laser confocal imaging showed that IS promoted the translocation of NF-κB-p65 from cytoplasm to nucleus.Further intervention with NAC showed that NAC partially reversed the high phosphorylation levels of p38 MAPK and p44/42 MAPK induced by IS,but it had no effect on the high phosphorylation levels of NF-κB induced by IS.9.After pretreatment of NRVM with p38MAPK inhibitor SB203580,it can be seen that SB203580 can partially reverse the down-regulation of Kv4.2 and Kv4.3 mRNA and protein induced by IS,but it has no effect on the down-regulation of KChIP2 mRNA and protein induced by IS.10.SB203580 can partially reverse the decrease in Ito and f current induced by IS.When the command voltage is 60 mV,it can be seen that the current density of Ito,f in the IS+SB group(11.20±0.50)is significantly higher than that in the IS group(8.12±0.23).(P<0.05).In addition,SB203580 partially reversed the acceleration of activation and decay of Ito,f current induced by IS,and partially reversed the prolongation of action potential duration induced by IS.11.The p44/42 MAPK inhibitor U0126-EtOH partially reversed the down-regulation of Kv4.2,Kv4.3 and KChIP2 mRNA and protein induced by IS.12.When the command voltage is 60mV,it can be seen that the current density of Ito,f in the IS+U0126 group(11.52±0.59)is significantly higher than that in the IS group(8.00±0.33)(P<0.05).U0126-EtOH can partially reverse the acceleration of activation and decay of Ito,f current caused by IS,and partially reverse the prolongation of the action potential caused by IS.13.The NF-κB inhibitor BAY 11-7082 partially reversed the down-regulation of Kv4.3 and KChIP2 mRNA and protein induced by IS.14.When the command voltage is 60mV,the current density of Ito,f in the IS+BAY group is 11.11 ±0.30,which shows that its value is significantly higher than that in the IS group(8.27±0.23)(P<0.05).In addition,BAY 11-7082 can partially reverse the acceleration of activation and decay of Ito,f current caused by IS,and also reverse the prolongation of the action potential induced by IS.Conclusions:1.ROS can be effectively activated by IS in NRVM,and NOX2 protein is a potential source of ROS.2.Indoxyl sulfate reduced the expression levels of Kv4.2,Kv4.3 and KChIP2 mRNA and protein in NRVM by activating ROS.3.Indoxyl sulfate reduces the current density of Ito,f by activating ROS,accelerates the activation and decay of Ito,f current,and prolongs the duration of the action potential.4.Indoxyl sulfate indirectly increases the phosphorylation level of p38 MAPK and p44/42 MAPK by activating ROS.In addition,it also directly increases the phosphorylation level of NF-κB.5.Indoxyl sulfate down-regulates the expression of Kv4.2 and Kv4.3 mRNA and protein by increasing p38 MAPK phosphorylation,thereby reducing the Ito,f current density,accelerating the activation and decay of Ito,f current,and prolonging the action potential duration.6.Indoxyl sulfate down-regulated the expression levels of Kv4.2,Kv4.3 and KChIP2 mRNA and protein in NRVM by increasing p44/42 MAPK phosphorylation,thereby reducing the Ito,f current density,accelerating the activation and decay of Ito,f current and prolonging the action potential duration.7.Indoxyl sulfate down-regulates the expression of Kv4.3 and KChIP2 mRNA and protein by increasing NF-κB phosphorylation,thereby reducing the Ito,f current density,accelerating the activation and attenuation of Ito,f current,and prolonging the action potential duration.8.Indoxyl sulfate reduces the expression of Ito,f current-related mRNA and protein by activating ROS/p38 MAPK,ROS/p44/42 MAPK and NF-kB signaling pathways,which successively causes the decrease of Ito,f current and prolonged action potential duration.Part Ⅲ Indoxyl sulfate increases the susceptibility of CKD rats to ventricular arrhythmiaObjective:To determine the effect of indoxyl sulfate on the susceptibility of arrhythmia in rats with chronic kidney diseasechronic kidney disease(CKD).Methods:Twenty-seven male wistar rats were selected and divided into Sham group,CKD group,CKD+BB536 group,Vehicle group and IS group.Then the ELISA method was used to detect the expression of indoxyl sulfate in rat serum and heart tissue in each group.The protein expression levels of Kv4.2,Kv4.3、KChIP2、NOX2、Phospho-p38 MAPK、p38 MAPK、Phospho-p44/42MAPK(Erk1/2)、p44/42 MAPK(Erk 1/2)、Phospho-NF-κBp65 和 NF-κB-p65 in rat hearts in each group were detected by Western blot.DHE was used to stain frozen sections of rat hearts to detect the expression of ROS.Finally,in the RM6240B/C multi-channel physiological signal acquisition and processing system,the limb lead Ⅱ configuration was used to record the rat’s ECG.In order to detect the susceptibility of rats to arrhythmia,rats of different groups were injected intraperitoneally with isoproterenol and caffeine to stimulate the arrhythmia,and the induced arrhythmia was scored and quantified.Results:1.Serum IS expression level in CKD group was significantly higher than that in Sham group(P<0.01),while serum IS expression level in CKD+BB536 group was significantly lower than that in CKD group(P<0.01),The serum IS expression level of the IS group was significantly higher than that of the Vehicle group(P<0.01),and the change of IS expression level in rat heart tissue also showed similar results.2.The expression levels of Kv4.2,Kv4.3 and KChIP2 protein in the myocardium of the CKD group were significantly lower than those in the Sham group(all P values<0.01),while the Kv4.2,Kv4.3 and KChIP2 proteins in the CKD+BB536 group The expression levels were significantly higher than those in the CKD group(all P values<0.05).3.The relative expression levels of Kv4.2,Kv4.3 and KChIP2 proteins in the rat myocardium in the IS group were significantly lower than those in the Vehicle group(all P values<0.01).4.The relative DHE fluorescence intensity in the myocardium of the CKD group was significantly higher than that in the Sham group(P<0.01),while the DHE fluorescence intensity in the CKD+BB536 group was significantly weaker than that in the CKD group(P<0.01).The fluorescence intensity of DHE in the IS group was significantly higher than that in the Vehicle group(P<0.01).5.The relative expression of NOX2 protein in the myocardium of rats in the CKD group was significantly higher than that of Sham(P<0.01),while the expression of NOX2 protein in the CKD+BB536 group was significantly lower than that in the CKD group(P<0.01).The relative expression of NOX2 protein in IS group was significantly higher than that in Vehicle group(P<0.01).6.The phosphorylation levels of p38 MAPK,p44/42 MAPK and NF-κB protein in the rat myocardium in the CKD group were significantly higher than those in the Sham group.The phosphorylation level of p38 MAPK,p44/42 MAPK and NF-κB protein in the CKD+BB536 group was significantly lower than that in the CKD group.Similarly,the phosphorylation levels of p38 MAPK,p44/42 MAPK and NF-κB proteins in the IS group were also significantly higher than those in the Vehicle group.7.The QT interval value in the CKD group(123.60±1.17)was significantly higher than that in the Sham group(73.00±0.89)(P<0.01),while the QT interval value in the CKD+BB536 group(91.67±1.09)was significantly lower than that in the CKD group Small(P<0.01).The QT interval value in the IS group(107.20±2.54)was significantly larger than that in the Vehicle group(81.50±1.38)(P<0.01).8.After intraperitoneal injection of isoproterenol and caffeine,Ventricular tachycardia was observed in rats in the CKD group and IS group.After calculating the arrhythmia score,it was found that the arrhythmia score in the CKD group(3.80±0.20)was significantly higher than that in the Sham group(0.80±0.20)(P<0.01),while the arrhythmia score in the CKD+BB536 group(2.33±0.42)The score was significantly lower than that of the CKD group(P<0.05).The arrhythmia score in the IS group was also significantly higher than that in the Vehicle group(P<0.01).Conclusions:1.The expression levels of IS were significantly increased in the serum and myocardial tissue of CKD rats and IS-treated rats,respectively.2.The elevated IS in the myocardial tissue of CKD rats can effectively reduce the expression of Kv4.2,Kv4.3 and KChIP2 proteins in the myocardium.3.The elevated IS in the myocardial tissue of CKD rats activates ROS,NOX2 protein and participates in the production of ROS in the myocardium of CKD rats.4.The elevated IS in the heart of CKD rats activated the p38 MAPK,p44/42 MAPK and NF-κB signaling pathways in the rat myocardium.5.The elevated IS in CKD rats prolonged the QT interval.6.The elevated IS in CKD rats increases the susceptibility of CKD rats to ventricular arrhythmia. |
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