| Objective: At present,the acute blockade effects of amitriptyline and other antidepressants on sodium channel in cardiac myocytes have been reported,but it can not explain the common clinical long-term action of drugs causing Brugada syndrome(Br S)and other ventricular arrhythmias.To investigate the acute and chronic effects of amitriptyline on the sodium channel in ventricular myocytes and reveal the mechanism of drug-induced Br S.Methods: The effects of acute administration of amitriptyline for 1 day and chronic administration for 7 days on sodium channel in ventricular myocytes were compared.The influences of different duration of medication on ECG of rats were observed.Whole cell patch clamp technique was used to observe the sodium channel gating characteristics such as peak sodium current density,steady-state activation curve,steady-state inactivation curve and recovery curve after inactivation.The sodium channel gating characteristics were observed by attached patch on the intercalated disc and lateral membrane.The differences of the expression of sodium channel between the two groups were analyzed by western blotting.Confocal microscopy was used to observe the distribution of sodium channels in ventricular myocytes.Results:(1)The ST segment part of ECG in amitriptyline 1day of medicine group increased,and accounted for about 60%,and that of 7days of medicine group increased to 80%,and the increase amplitude was significantly higher than that in 1day group(0.3 ±0.07 m V vs 0.1± 0.09 m V,P< 0.05).(2)It was known through using whole cell patch clamp technique that the peak sodium current density of 1day amitriptyline medicine group reduced by 23% compared to the control group(-21.21 ± 0.35 p A/p F vs-30.24 ± 2.62 p A/p F,P<0.01).The peak sodium current density of 7d of medicine group reduced by 54% compared to control group(-14.63 ± 0.75 p A/p F vs-30.24 ± 2.62 p A/p F,P<0.01).With the whole cell patch clamp technique,it could be known that the ? of 1day of medicine group extended from 92.68 ± 34.48 ms to 135.56 ± 6.87 ms(P<0.05),and the ? of 7days of medicine group extended from 92.68 ± 34.48 ms to 137.63 ± 9.32 ms(P<0.05).However,the medicine way of 1day and 7days had no differences in their change degree to recovery curves after sodium channel inactivation.(3)At lateral membrane,the 7days of medicine group decreased(-259.53±7.34 p A vs-128.76±8.52 p A,P<0.05)and was greater than that of 1day(-259.53±7.34 p A vs-166.76±7.24 p A,P<0.05).At intercalated disc,the descend range of 7days of medicine group(-666.34±15.85 p A vs-286.35±9.33 p A,P<0.05)was greater than that of 1day(-666.34±15.85 p A vs-456.13±10.65 p A,P<0.05),which suggested that chronic effect made the sodium current decrease obviously.At the intercalated disc sodium channel steady-state activation curve,the V1/2 channel of 1day of medicine group moved to the right from-22.54 ± 1.24 m V to-19.87 ± 1.67 m V(P<0.05),and that of 7days moved to the right from-22.54 ± 1.24 m V to 18.69± 2.33 m V(P<0.05).At the sodium channel inactivation recovery curve of side membrane,the extending degree of the recovery time constant(?)in the channel of 7days of medicine group(230.43 ± 8.98 ms vs 320.13 ± 38.22 ms,P < 0.05)was similar as that of 1day(230.43 ± 8.98 ms vs 324.47 ± 20.67 ms,P < 0.05).At the intercalated disc,the extending degree of the recovery time constant(?)in the channel of 7days of medicine group(210.57 ± 26.58 ms vs 352.50 ± 42.32 ms,P < 0.05)was similar as that of 1day(210.57 ± 26.58 ms vs 357.34 ± 39.69 ms,P < 0.05).The two medicine ways made the inactivation recovery time of intercalated disc and lateral membrane promote significantly.(4)The expression of membrane channel in amitriptyline reduced(1day:1.04 ± 0.07 vs.0.73 ± 0.07,P < 0.05;7days:1.04 ± 0.07 vs 0.544 ± 0.08,P < 0.05),and 7days of medicine group was most obvious.(5)Compared with amitriptyline 1day of medicine group,the red fluorescence density decreased more significantly in the lateral membrane and the intercalated disc of the 7days of medicine group,and the cytoplasm had granular or small flaps of fluorescent particles.Conclusion:(1)This study found that the acute effects of amitriptyline(1 day)and the chronic effects of long-term administration(7 days)had a blockade effect on sodium channels in rat ventricular myocytes,and the chronic effects of long-term use were more significant.(2)The effect of amitriptyline on the acute and chronic effects was similar on the Nav1.5 gating characteristics,and the recovery time after inactivation was prolonged,and the steady-state activation and steady-state inactivation curves showed no difference.(3)The chronic effect of amitriptyline showed to decrease the distribution of sodium channel protein on the ventricular myocytes membrane and the expression of the site of sacral disks of rat.(3)Compared with the acute effects of amitriptyline,chronic effects led to a more pronounced decrease in the expression of Nav1.5 protein in the cell membrane.But the two had similar effects on the gating characteristics of sodium channels.We considered that amitriptyline may affect the trafficing of sodium channels to the cell membrane and it provided a new idea for preventing di BrS. |
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