The Effect Of Daidzein On Ion Channels Of Rat Ventricular Myocytes

Objective:To observe the effects of daidzein(DD)on the current and kinetic characteristics of sodium channels,transient outward potassium channels and L-type calcium channels in rat ventricular myocytes,and to investigate the mechanism of its protective effect on cardiomyocytesMethods:MTT assay was used to detect the toxicity of DD.Langendorff aortic retrograde perfusion and single enzymatic dissolution were used to isolate single rat cardiomyocyte.By using whole cell patch clamp technique,to observe,record and analyze the changes of ion channel currents and their dynamic characteristics in rat cardiomyocytes before administration and low(1?mol/L),medium(3 ?mol/L)and high(10 ?mol/L)DD under voltage clamp mode.Results:1.Effects of DD on the viability of neonatal rat cardiomyocytes:Myocardial cells of SD rats were digested with 0.25%trypsin solution for 4 times to obtain cardiomyocytes.After inoculation in a Petri dish,change the solution every 24-48 h.When 80-90%of the myocardial cells were observed under inverted microscope,DD(0.1,0.3,1,3,10,30,100)?mol/L was added to the cultured myocardial cells of neonatal rats.After 24 hours of further culture,the survival rate of the cells decreased with the increase of drug concentration.Among them,when the final concentration of DD was 30-100 ?mol/L,the decrease of myocardial cell viability was the most obvious,and the difference was significant compared with that control group(P<0.05)Therefore,The subsequent patch clamp experiments were performed with DD of 1,3 and 10?mol/L.2.Morphological observation of isolated myocardial cardiomyocytes:Acutely isolated single cardiomyocytes were observed under an inverted microscope.The first isolated rat cardiomyocytes were mostly rod-shaped,with clear horizontal stripes,complete cell membrane edges,strong stereoscopic effect,and no depressions or bubbles on the surface.Most of them were in a resting state(70%-90%),and a few of them have contracted to ellipsoid or globular shape.The survival of cardiomyocytes requires energy and oxygen,and Ca2+ regulated its contraction and relaxation.After recalcification,40%-60%of the cells remained in the original form,which will be used in the following drug intervention experiments.3.Effect of DD on sodium channel in rat cardiomyocytes:(1)Concentration dependence of DD on INa:INa was guided by the agreed holding potential and clamping potential.Compared with the control group,low concentration(DD 0.1 umol/L)had almost no inhibitory effect on INa,while DD(0.3-10 mol/L)inhibited INa in a concentration-dependent manner.INa current density decreased from(-53.9±6.35)pA/pF(DD 0 umol/L)to(-33.78±3.24)pA/pF,(-25.16±4.24)pA/pF,(-22.28±3.54)pA/pF and(-17.96±4.56)pA/pF(P<0.05),Which inhibited current density in a concentration-dependent manner.(2)The current-voltage(I-V)curve of DD to INa:Compared with the control group,DD 1,3,and 10 ?mol/L caused the current-voltage(I-V)curve to move upward.However,the trajectory trend of ?-? curve,the activation potential of the channel current,peak current potential(Vpeak)and reversal potential(VRev)of channel current remained unchanged.(3)Activation and inactivation kinetics of INa by DD:The activation and inactivation curves of INa were guided according to specific stimulation schemes.The effects of different concentrations of DD(1,3,10 ?mol/L)on the activation and inactivation curves of INa were observed.The results showed that DD(1,3,10 ?mol/L)made the activation curve move towards depolarization.The half activation voltage V1/2 of the activation curve before and at the three concentrations were(-40.99±0.32),(-38.81±0.19)mV,(-35.67±0.78)mV,(-31.94±0.43)mV(P>0.05),which showed that DD delayed the activation of sodium channel.DD(1,3,10 mol/L)makes the inactivation curve move towards hyperpolarization.The half inactivation voltage V1/2 of inactivation curve before administration and at three concentrations were(-74.42±0.44)mV,(-79.74±0.45)mV,(-82.81±0.59)mV and(-86.5±0,77)mV(P<0.05),it suggested that DD accelerated the inactivation of sodium channel.(4)Recovery curve of DD after inactivation of INa:Under the same conditions,after DD administration,the recovery curve of INa was shifted to the left,T was the recovery time constant after channel inactivation,which changed from(21.35 ± 0.13)ms before administration to(15.49±0.24)ms,(13.30±0.32)ms ?(11.31±0.22)ms(P<0.05).The results showed that DD accelerated the transition time of INa from inactivation to activation in a concentration-dependent manner.4.Effects of DD on transient outward potassium channels in rat cardiomyocytes:(1)Concentration dependence of DD on Ito:The steady-state activation program induced an outward current,which can be significantly blocked by adding 4-AP,which confirmed that the current is Ito.When the current tends to stabilize,the Ito gradually returned to its pre-administration level after washing with extracellular fluid,suggesting that the effect of DD on Ito was reversible.DD(0.1 ?mol/L)had little effect on Ito,but DD had stronger and stronger inhibitory effect on Ito of ventricular myocytes at 0.3,1,3 and 10 ?mol/L.(2)The current-voltage(I-V)curve of DD to Ito:Under DD(0-10 ?mol/L),The program was given continuous changing steps and measured the channel current to make Ito current-voltage(I-V)curve.Compared with the control group,the current density of Ito decreased with the increase of concentration.However,the shape of ?-? curve and reversal potential remained unchanged.(3)Activation and inactivation kinetics of Ito by DD:Under the blank control condition,the V1/2 of normalized fitting activation conductivity curve was(13.07±0.58)mV and k was(13.87±0.52).1,3 and 10 ?mol/L DD were(15.76±0.40)mV/(13.43 ±0.35),(17.59±0.67)mV/(13.83±0.59),(19.65±0.48)mV/(13.34±0.43)respectively(P>0.05).The V1/2 and k values of the steady-state inactivation curves DD(0,1,3,10 ?mol/L)were(-20.03 ± 0.41)mV/(-15.16±0.36),(-22.27 ± 0.35)mV/(15.50±0.31),(-24.89±0.42)mV/(15.28 ± 0.38),(-27.74±0.55)mV/(15.31±0.49)(P>0.05).When DD acted,the steady-state and activation curves hardly moved.Therefore,DD did not affect the opening and closing of channels from the activation and inactivation of Ito.(4)Recovery curve of DD after inactivation of Ito:When the cell membrane voltage was stimulated,the open channel gradually inactivated,and then the voltage stimulation channel would no longer open to inactivation.Finally,a voltage stimulation was given,and the inactivation gradually recovered.The current was restored after the transient outward potassium channel was inactivated before and after the DD,and the current recovery curve was fitted.DD caused the recovery curve to shift to the right after Ito was inactivated,and the ? value prolonged with increasing concentration(P<0.05).5.Effect of DD on L-type calcium channel in rat cardiomyocytes:(1)Determination of Ica-L in Myocardial Cells:A series of inward currents were induced by square wave stimulation.These currents were almost blocked,almost not blocked and almost blocked by Tyrode's solution perfusion containing CdCl2(calcium channel blocker),NiCl2(specific T-type calcium channel blocker)and verapamil(specific L-type calcium channel blocker),respectively.It was proved that the recorded current was L-type calcium current(P<0.01).DD(3 ?mol/L)significantly inhibited Ica-L,peak(P<0.01).(2)Concentration dependence of DD on ICa-L:To investigate the effect of DD on ICa-L in ventricular myocytes at concentrations of 0.1,0.3,1,3,and 10 ?mol/L.DD(0.1 ?mol/L)had a little effect on ICa-L,while DD(0.3,1,3,10 ?mol/L)caused ICa-L in rat ventricular myocytes from pre-dose(-17.39±0.61)pA/pF decreased to(-11.44±0.67)pA/pF,(-9.53±1.58)pA/pF,(-6.70±0.65)pA/pF and(-2.45±0.54)pA/pF,respectively,Compared with the control group,DD at low concentration of 0.3 and 1 ?mol/L had obvious inhibitory effect(P<0.05),suggesting that DD inhibited L-type calcium channel current in a concentration-dependent manner,and the stronger the inhibitory effect was with the increase of DD concentration.When the current tends to stabilize,the ICa-L current gradually returns to its pre-administration level after washing with extracellular fluid,suggesting that the effect of DD on ICa-L was reversible.(3)The current-voltage(I-V)curve of DD to Ica-L:When DD(1-10 ?mol/L)acted,Ica-L and current density decreased gradually.The ?-? curve moved up significantly with the decrease of current density in the voltage range(-20-+20 mV).The shape of ?-? curve and inversion potential did not change significantly.(4)Activation and inactivation kinetics of ICa-L:The activation curve and inactivation curve of Ica-L move to the direction of hyperpolarization with the increase of DD concentration.The half-maximal activation voltage V1/2 and slope factor k of the activation curve before administration and DD(1-10 ?mol/L)were(-9.13±0.57)mV/(4.52±0.55),(-14.10±0.65)mV/(4.70±0.54),(-20.17±1.55)mV/(5.00±1.44),(-28.76±1.44)mV/(4.80±1.33)(P<0.05).It is suggested that DD can accelerate the activation of L-type calcium channel.The half-maximal inactivation voltage V1/2 and slope factor k before the administration and the inactivation curve of DD were(-19.82±0.55)mV/(14,11±0.49),(-30.21±0.70)mV/(16.29±0.63),(-42.61±1.00)mV/(15.77±0.92)and(-52.83±1.16)mV/(12.66±1.08)(P<0.05).(5)Recovery curve of DD after inactivation of ICa-L:DD shifted the recovery curve after inactivation of ICa-Lto the right,while 1 ?mol/L DD,? changed from(10,50±0.45)ms to(16.75±0 65)ms before administration,and after 3 and 10 ?mol/L DD,? was(21.50±0.73)ms and(29 00±1.07)ms,and recovery time was significantly prolonged(P<0.05).The results showed that DD prolonged the transition time of Ica-L from inactivation to activation in a concentration-dependent manner.Conclusion:DD blocks the transient outward potassium channel in a concentration-dependent manner;both sodium channel and L-type calcium channel can be concentration-dependent and change the kinetic characteristic block.These results may be the mechanism of action of DD to protect heart.