| Objective:To investigate the effects of saffron acid CA(crocetin acid)on the electrocardiogram of rats with atrial fibrillation(AF)induced by calcium chloride-acetylcholine(Ach-CaCl2)in vivo,and to explore the main ion channels on cardiac myocytes and their kinetic characteristics from an electrophysiological perspective,and to elucidate the relationship between the anti-drug-induced AF effect of saffron acid and each ion channelMethods:1.The electrocardiogram(ECG)was recorded with standard Ⅱ-lead guidance to observe the effect of CA on the ECG of rats with AF induced in vivo by Ach-CaCl2 administration method.2.The effect of CA on cardiomyocyte viability in SD mammary rats was detected using MTT method.3.Aortic reverse perfusion was performed in isolated rat hearts using the langendorff device,and type Ⅱ collagenase with gradient recalcification was used to isolate calcium-resistant stable individual cardiomyocytes.4.Whole-cell membrane clamp technique was applied to guide the recording,observation and analysis of the effects of different concentrations of CA on several major ion channel currents and their kinetic characteristics on rat cardiac myocytes.Results:1.Effects of CA on the ECG of AF model ratsWe established a drug-induced AF model by tail vein injection of Ach-CaCl2 in rats,and observed the changes of ECG in different groups with different doses of CA prophylactic administration.The results showed that compared with the model group,the evoked time of AF changed from(4.33 ± 1.21)s to(5.32 ± 0.85)s(low dose group)(n=6,p>0.05),(6.64± 1.05)s(medium dose group)(n=6,p<0.01),(7.50 ± 0.66)s(high dose group)(n=6,p<0.01),respectively,while the duration of AF changed from(7.83 ± 1.02)s to(6.48± 1.25)s(low dose group)(n=6,p>0.05)(5.78± 1.58)s(medium dose group),(4.53± 1.26)s(high dose group)(n=6,p<0.05).(n=6,p<0.05).It is evident that CA can significantly improve the ECG abnormalities in calcium chloride-acetylcholine-induced AF rats,and different doses of CA can prolong the AF induction time and reduce the AF duration in model animals compared with the control group.2.Effects of different concentrations of CA on cell viability of SD mammary ratsThe effect of CA at different concentrations(25,50,100,and 200 μmol·L-1)on cardiomyocyte activity in SD mammary rats was examined by MTT method,and the results showed that the inhibition of cell activity by CA was(6.70 ± 3.19)%,(17.17±2.16)%,(19.98 ± 0.81)%(P>0.05),(31.21 ± 1.80)%(P<0.05).Regarding the kinetic characteristics of Ito:after administration of 50,100,and 200 μmol·L-1 of CA,the half-activation potential(V1/2-a)increased from(4.94 ± 0.45)mV before administration to(10.61± 0.63)mV,(19.55 ± 0.87)mV,and(22.85± 0.67)mV,respectively(P<0.01,n=7).It indicates that the CA blocking channel enters the activated state from the resting state,significantly increasing the difficulty of activation to Ito.The recovery time constant(τ value)increased from(12.06 ± 1.09)ms before administration to(14.17 ± 0.62)ms(P>0.05,n=7),(17.22± 0.83)ms,and(21.30 ± 2.95)ms(P<0.01,n=7),respectively,prolonging the transition from the inactivated to the activated state of Ito.CA also shifted the Ⅰ-Ⅴ curve gradually upward,but the overall trend of movement and morphology did not change much.According to the above changes,it is speculated that CA further reduces the incidence of AF by inhibiting Ito,prolonging the duration of action potentials,and reducing the possibility of channels being activated in the resting state.3.Effects of CA on transient outward potassium channels in rat atrial myocytesWe used a whole-cell membrane clamp technique to guide and record transient outward potassium channels and showed that Ito was significantly inhibited in rat atrial myocytes at CA doses≥ 50 μmol·L-1.CA at 50,100,and 200 μmol·L-1 was able to reduce the peak Ito from(71.10 ± 7.28)pA/pF before administration to(55.49 ± 2.34)pA/pF,(35.56 ± 7.57)pA/pF and(14.69 ± 1.95)pA/pF(n=7,P<0.01).With respect to the kinetic characteristics of Ito:after administration of 50,100 and 200 μmol·L-1 CA,the half-activation potential(V1/2-a)increased from(4.94 ± 0.45)mV before administration to(10.61 ± 0.63)mV,(19.55± 0.87)mV,and(22.85±0.67)mV,respectively(P<0.01,n=7).This indicates that CA blocking channels enter the activated state from the resting state,significantly increasing the difficulty of activation of Ito;the half-inactivation potential(V1/2-in)also decreased from(-23.01 ± 1.14)mV before drug administration to(-24.84 ± 1.27)mV(n=7,P>0.05),(-29.42 ± 1.75)mV and(-34.72±1.82)mV(n=7,P<0.01),which decreased V1/-in and shifted the inactivation curve to the left;the recovery time constant(τ value)also increased from(12.06±1.09)ms before administration to(14.17±0.62)ms(P>0.05,n=7),(17.22 ±0.83)ms,and(21.30±2.95)ms(P<0.01,n=7),which prolonged the transition from the inactivated to the activated state of Ito.CA also shifted the Ⅰ-Ⅴ curve upward,but the overall trend and shape of the shift did not change much.4.Effects of CA on L-type calcium channels in rat atrial myocytesThe inward current ICa-L was guided and recorded using the voltage clamp mode with the corresponding stimulation protocol,and the effect of each dose of CA on the current was observed using the self-control method before and after administration.The results showed that 50,100,and 200 pmol·L-1 of CA were able to reduce the peak ICa-L current from(-31.37± 3.35)pA/pF before administration to(-25.18 ± 3.39)pA/pF(n=7,P<0.05),(-21.79 ±4.02)pA/pF,and(-18.69±4.87)pA/pF(n=7,P<0.01).Regarding the kinetic characteristics of Ica-L,the current-voltage(I-V)relationship curves of ICa-L were significantly shifted upward by different concentrations of CA,while the trend of the curves,activation potentials and peak potentials were largely unaffected;the activation curves were shifted to the right in the direction of the positive axis,and 100 and 200 μmol·L-1 CA shifted the half-activation voltage(V1/2-ac)from(-31.24 ± 2.05)before administration to(-18.94 ±2.24)mV and(-5.59 ± 2.41)mV,respectively(n=7,P<0.01).It showed that CA improved the ease of L-type calcium channel opening;CA significantly shifted the inactivation curve to the left,and the half inactivation voltage(V1/2-in)decreased from(-20.89±0.80)mV before administration to(-30.04 ± 1.08)mV,(-52.75 ± 1.32)mV after the addition of 100 and 200 μmol·L-1 of CA(n=7,P<0.01).The slope factor κ changed sequentially to(-12.04 ± 0.96),(-10.61 ± 1.19),implying that CA accelerated the shift of L-type calcium channels from the activated to the inactivated state;CA at 100 and 200 μmol·L-1 shifted the post-inactivation recovery curve to the right,and the recovery time τ increased from(13.94 ±1.24)ms before administration to(20.55 ± 1.29)ms and(27.85 ± 1.49)ms,respectively(n=7,P<0.01).5.Effects of CA on sodium channels in rat atrial myocytesWe used a whole-cell membrane clamp technique to guide and record the voltage-gated sodium channel current INa,and observed the changes in this current at each dose of CA using a self-control method before and after administration.The results showed that CA at 50,100,and 200 μmol·L-1 was able to reduce the peak INa current from(-83.83±3.66)pA/pF before administration to(-56.44 ± 3.16)pA/pF,(-45.14± 3.74)pA/pF,and(-32.89 ±3.90)pA/pF,respectively(n=7,P<0.01).In terms of kinetic characteristics:CA at 100 and 200 μmol·L-1 both shifted the current-voltage(I-V)relationship curve of INa upward,but the trend of the curve,activation potential and peak potential were basically unaffected;it shifted the activation curve to the right,i.e.in the direction of depolarization.After adding 100 and 200 μmol·L-1 CA,the half activation voltage(Vi/2-ac)became larger from(-58.77 ±0.39)mV before drug administration to(54.93 ± 0.35)mV and(-51.89±0.25)mV,respectively(n=7,P<0.01),i.e.,CA improved the ease of voltage-gated sodium channel opening;it could make INa deactivation mechanics plot to the left,i.e.,shifted toward hyperpolarization.The half inactivation voltage(V1/2-in)decreased from(-77.35± 0.49)mV before the addition of the drug to(-81.16 ± 0.54)mV and(-85.32 ± 0.61)mV(n=7,P<0.01)after the addition of 100 and 200 μmol·L-1 of CA(n=7,P<0.01).The slope factor κchanged sequentially from(-10.23 ± 0.44)to(-9.05 ± 0.47)and(-8.44±0.54)before administration(n=7,P<0.01),and CA accelerated the transition from the activated to the inactivated state of voltage-gated sodium channels;CA also affected the post-inactivation recovery process of INa:CA at 100 and 200 μmol·L-1 shifted the recovery curve of sodium channels to the right along the time axis.CA also affected the post-inactivation recovery process of INa:100 and 200 μmol·L-1 CA shifted the post-inactivation recovery curve to the right along the time axis,and the recovery time τ increased from(20.98±1.27)ms to(31.86± 2.15)ms and(45.27 ± 7.72)ms,respectively,before the administration of CA(n=7,P<0.01).Conclusions:CA pretreatment antagonized calcium chloride-acetylcholine-induced paroxysmal atrial fibrillation in rats,and this effect was associated with inhibition of Ito,ICa-L,and INa and alteration of their channel kinetic properties. |
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