心肌细胞感染病毒后钙、钾通道电流及其表达改变和牛磺酸的作用

The effects of taurine on ion currents and their expression in cardiomyocytes after CVB3 infection

目的 观察心肌细胞感染病毒后心肌细胞钙、钾通道电流及通道表达改变和牛磺酸的作用。方法 (1 )体外心肌细胞感染病毒 :用胰蛋白酶和胶原酶消化法分别获得大鼠、乳鼠培养和分离的心肌细胞并感染柯萨奇B3病毒 ,用于检测Ca2 +跨膜内流、电压依赖性钙通道电流 (ICa)和外向钾通道电流 (Iout)。 (2 )在体小鼠感染病毒 :BALB c小鼠腹腔内注射柯萨奇B3病毒 7d后取出心脏用于检测电压依赖性钙通道α1 亚单位抗原和电压调控性钾通道 (Kv)mRNA的表达。相应各组加入不同剂量的牛磺酸并观察其作用。结果 (1 )感染柯萨奇B3病毒后Ca2 +跨膜内流量及ICa增加 ,牛磺酸对其有抑制作用。 (2 )柯萨奇B3病毒感染使Iout明显增大 ,牛磺酸可使病毒感染后的Iout趋于恢复正常。(3)柯萨奇B3病毒感染后小鼠心肌出现强电压依赖性钙通道α1 亚基抗原阳性信号 ,Kv1 2、Kv2 1、Kv4 2mRNA与 3种探针的杂交信号亦明显增强 ,牛磺酸可使增强的阳性信号减弱。结论 柯萨奇B3病毒感染后心肌细胞钙、钾离子通道电流及表达均增加 。

Objective To investigate the effects of taurine on current of L type voltage dependent calcium channel (I Ca ), outward potassium current (I out ) and their expression in cardiomyocytes after coxsackievirus B3 (CVB3) infection Methods (1)Heart cell preparation and virus infection: monolayer culture heart cells were made from newborn SD rats hearts by 5 cycles of enzymatic digestion with trypsin solution and were used for detecting calcium influx across membrane (by liquid scintillating techniques and radioactive isotope 45 Ca 2+ ) Cardiomyocytes from the newborn SD rats were obtained using collagenase digestion and used for recording outward potassium current (I out ) (by Hamill patch clamp methods) Heart from adult SD rats was put to Langendoff installation and perfused by collagenase to obtain single cardiomyocytes, which was used for recording current of L type voltage I Ca . One hundred to two hundred times of tissue culture infections dose 50 (TCID 50 ) of Coxsackie virus B3 (CVB3) were added to the medium for infection the heart cells (2) Infect BALB/c rats by CVB3: to set up the whole body viral myocarditis models, CVB3 MEM eagle′s solution was injected through intraperitoneal to 4 weeks old BALB/c rats and the hearts were removed after 7 days′ infection for investigating the expression of voltage gate potassium channel (Kv) mRNA (in situ hybridization) and LVDCC α 1 subunit (immunohistochemical methods) All experiments were divided into 3 groups: control group (C), virus infection group (V) and taurine treated group (V+T), in which different dose of taurine was applied Results (1) Taurine could inhibit the Ca 2+ influx at high extracellular calcium concentration in the normal cardiomyocytes and the similar effect could be seen in I Ca Calcium influx and I Ca were increased after CVB3 infection ( P <0 05) and taurine could attenuate the changes and made the membrane potential depolarizing to peak current restored to normal (2) Taurine could thwart the enhancement of I out after virus infection ( P <0 05) but taurine had no effect on I out in normal rat cardiomyocytes (3) The index of positive hybridization signals of Kv mRNA was increased after CVB3 infection ( P <0 01) and taurine could lessen the increased expression level ( P <0 05) (4) The LVDCC α 1 subunit (antigen) was much reinforced after CVB3 infection and could be decreased by taurine treatment ( P <0 05) Conclusion The ion currents and expression levels of potassium and calcium channel were increased after CVB3 infection The beneficial effect of taurine on viral myocarditis might be through the inhibition of these changes