背景:电压门控钾(Kv)通道的生长与心律失常的发展密切相关。Salidroside(Sal),一种来自大花红景天的活性成分,已被证明对心脏病有保护作用。本研究旨在研究Sal对Kv2.1通道的影响,并探讨了抗心律失常的离子机制。
方法:在本研究中,我们利用了西沙必利(顺式。,延长QT间期并引起心律失常的兴奋剂)通过静脉注射建立心律失常模型,并检测了Sal对SD大鼠心电图(ECG)和压力容量环(P-Vloop)的影响。Sal对西酞普兰心电图的影响(Cit。,aKv2通道抑制)诱发心律失常大鼠模型通过监测心电图多项指标的动态变化进一步评价。然后,我们使用CCK-8法检测了Sal对缺氧H9c2细胞活力的影响。之后,使用全细胞膜片钳技术检查了Sal在正常和低氧条件下对H9c2细胞中Kv通道电流(IKv)和Kv2.1通道电流(IKv2.1)的影响。此外,在Kv和Kv2.1通道的抑制下测定Sal对H9c2细胞中IKv和IKv2.1的影响。用Kv2.1质粒稳定转染的HEK293细胞也用于研究在Sal预处理和共孵育条件下的IKv2.1变化。此外,通过分子对接预测和测试Sal与Kv2.1蛋白的潜在相互作用,分子动力学模拟(MDS),局域表面等离子体共振(LSPR),和细胞热转移测定(CETSA)技术,分别。此外,通过qRT-PCR估计Sal处理的H9c2细胞中Kv2.1的基因和蛋白质水平,蛋白质印迹(WB)和免疫荧光(IF)分析。
结果:Sal缩短了Cis引起的SD大鼠QT间期延长,改善了与心律失常相关的心脏损害。,如ECG和P-V环路数据所反映。Sal还对Kv2通道抑制引起的大鼠心律失常具有保护作用。在细胞层面,在CoCl2诱导的H9c2细胞缺氧损伤后,Sal增加了细胞活力。全细胞膜片钳实验证实,Sal在正常H9c2细胞中同时抑制IKv和IKv2.1,同时增强缺氧损伤后心肌细胞的IKv和IKv2.1。Sal增强了1.5mM4-AP抑制的IKv,并上调了20mM4-AP诱导的Kv2通道的所有抑制,拮抗Cit的IKv2.1抑制作用。此外,Sal预施用24小时和立即施用增加了用Kv2.1质粒稳定转染的HEK293细胞中的IKv2.1。分子对接证明了Sal与Kv2.1蛋白的潜在结合,计算的结合能为-5.4kcal/mol。MDS试验表明,Sal-Kv2.1配合物的平均氢键为30.89%。LSPR结果验证了Sal与Kv2.1蛋白的潜在结合,亲和力值为9.95×10-4M。CETSA测定证实Sal可以增强热处理的H9c2细胞中Kv2.1蛋白的表达,这表明Sal可能与Kv2.1蛋白结合。WB的结果,qRT-PCR,和IF进一步认为,Sal预施用24小时可增强Kv2.1基因和蛋白质的水平(对与Sal共孵育6小时和12小时的H9c2细胞的Kv2.1基因和蛋白质没有影响)。
结论:总体而言,我们的发现表明Sal可以抵抗SD大鼠的药物引起的心律失常,部分通过刺激Kv2.1调节复极化。
BACKGROUND: Voltage-gated potassium (Kv) channel growth is strongly associated with the development of arrhythmia. Salidroside (Sal), an active component from Rhodiola crenulata, has been shown to exert protective effects against heart disease. The present study was conducted to investigate the effects of Sal on Kv2.1 channel, and to explore the ionic mechanism of anti-arrhythmic.
METHODS: In this study, we utilized cisapride (Cis., A stimulant that prolongs the QT interval and causes cardiac arrhythmias) by intravenous injection to establish an arrhythmia model, and detected the effects of Sal on electrocardiography (ECG) and pressure volume loop (P-V loop) in SD rats. The effect of Sal on ECG of citalopram (Cit., a Kv2 channel inhibition)-evoked arrhythmia rat models was further evaluated by monitoring the dynamic changes of multiple indicators of ECG. Then, we detected the effect of Sal on the viability of hypoxic H9c2 cells using CCK-8 assay. After that, the effect of Sal on Kv channel currents (IKv) and Kv2.1 channel currents (IKv2.1) in H9c2 cells under normal and hypoxic conditions was examined using whole-cell patch clamp technique. In addition, the effect of Sal on IKv and IKv2.1 in H9c2 cells was determined under the inhibition of Kv and Kv2.1 channels. HEK293 cells stably transfected with Kv2.1 plasmids were also used to investigate the IKv2.1 changes under Sal pre-treated and co-incubated conditions. In addition, potential interactions of Sal with Kv2.1 protein were predicted and tested by molecular docking, molecular dynamics simulation (MDS), localized surface plasmon resonance (LSPR), and cellular thermal shift assay (CETSA) techniques, respectively. Furthermore, gene and protein levels of Kv2.1 in Sal-treated H9c2 cell were estimated by qRT-PCR, Western blot (WB) and immunofluorescence (IF) analysis.
RESULTS: Sal shortened the prolongated QT interval and ameliorated the cardiac impairment associated with arrhythmia in SD rats caused by Cis., as reflected in the ECG and P-V loop data. And Sal was also protective against arrhythmia in rats caused by Kv2 channel inhibition. At the cellular level, Sal increased cell viability after CoCl2-induced hypoxic injury in H9c2 cells. Whole-cell patch clamp assay confirmed that Sal inhibited both IKv and IKv2.1 in normal H9c2 cells, while enhanced IKv and IKv2.1 in cardiomyocytes after hypoxic injury. And Sal enhanced IKv inhibited by 1.5 mM 4-AP and upregulated all inhibition of Kv2 channels induced by 20 mM 4-AP administration, antagonized the IKv2.1 inhibitory effect of Cit. Moreover, Sal pre-administration for 24 h and immediate administration increased IKv2.1 in HEK293 cells stably transfected with Kv2.1 plasmids. Molecular docking demonstrated the potential binding of Sal to the Kv2.1 protein, with calculated binding energy of -5.4 kcal/mol. MDS test illustrated that the average hydrogen bonding of the Sal-Kv2.1 complexes was 30.89%. LSPR results verified the potential binding of Sal to Kv2.1 protein with an affinity value of 9.95 × 10-4 M. CETSA assay confirmed Sal can enhance the expression of Kv2.1 protein in H9c2 cells treated with heat, which suggests that Sal may bind to Kv2.1 protein. The results of WB, qRT-PCR, and IF further argued that Sal pre-administration for 24 h enhanced the levels of the Kv2.1 gene and protein (with no effects on the Kv2.1 gene and protein for H9c2 cells co-incubated with Sal for 6 h and 12 h).
CONCLUSIONS: Overall, our findings indicate that Sal can resist drug-induced arrhythmias in SD rats, partially by modulating repolarization through stimulating Kv2.1.