背景:槐定碱(SR)已显示出成为抗心律失常药的潜力。然而,SR的电生理特性和可药用性研究相对不足,这限制了SR作为抗心律失常候选药物的发展。
目的:为了促进SR作为抗心律失常候选药物的发展过程,我们对SR的体外和离体电生理特性进行了综合研究,以更全面地了解SR的多离子通道阻断效应,这为进一步的抗心律失常和安全性研究的可药用性研究奠定了基础。首先,通过全面整合膜片钳与电气和光学映射系统,在细胞和组织水平上记录并评估SR的电生理特性和抗心律失常电位。随后,SR的抗心律失常作用通过乌头碱和乌巴因诱导的心律失常在体内得到验证.最后,SR作为抗心律失常候选化合物的安全性是根据综合体外致心律失常试验(CiPA)指南进行评估的.
方法:在体外评估SR的抗心律失常作用,离体,和体内水平。
方法:制备分离的原代心肌细胞和稳定的细胞系,以通过全细胞膜片钳在体外探索作为多离子通道阻断剂的电生理特性。使用电气和光学测绘,在langendorff灌注的大鼠或豚鼠心脏中确定了SR的负变时效应。通过左冠状动脉结扎(LCAL)和异丙肾上腺素(ISO)诱导的离体快速性心律失常模型评估SR的抗心律失常活性。乌头碱和哇巴因诱发的心律失常的典型模型用于验证体内抗心律失常作用。最后,使用微电极阵列(MEA)在人诱导多能干细胞衍生心肌细胞(hSCCM)中检测到SR的致心律失常风险.
结果:单细胞贴片试验验证了SR在瞬态外向电流钾电流(Ito)中的多种离子通道阻断剂,l型钙电流(ICa-l),和快速激活延迟整流钾电流(IKr)。SR以浓度依赖性方式离体降低了心率(HR)和心室传导速度(CV),并延长了Q-T间隔。与HR的变化一致,SR延长了心脏的活动时间,并增加了在90%复极化(APD90)时测得的动作电位持续时间。在LCAL诱导的离体心律失常模型中,SR还可以显着延长发作时间并缩短自发性室性心动过速(VT)的持续时间。同时,在形成电交替和折返激发的ISO挑战之后,SR还可以显着上调编程的电刺激(PES)频率。此外,SR在乌头碱和乌巴因体内诱导的快速性心律失常模型中发挥了抗心律失常作用。值得注意的是,对于人类ether-à-go-go相关基因(hERG)通道的中度抑制,SR的致心律失常风险较低.此外,SR以浓度依赖性方式延长了hSCCM的场电位持续时间(FPDc),而不会在去极化(EAD)和心律失常发生。
结论:我们的结果表明,SR在电生理特性上表现为多种离子通道阻断剂,并在离体和体内发挥抗心律失常作用。同时,由于在hERG抑制试验和EAD的诱导中具有低的致心律失常风险,SR在室性快速性心律失常的治疗中具有巨大的潜力。
BACKGROUND: Sophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR\'s electrophysiological properties and druggability research are relatively inadequate, which limits the development of SR as an antiarrhythmic candidate.
OBJECTIVE: To facilitate the development process of SR as an antiarrhythmic candidate, we performed integrated studies on the electrophysiological properties of SR in vitro and ex vivo to gain more comprehensive insights into the multi-ion channel blocking effects of SR, which provided the foundation for the further drugability studies in antiarrhythmic and safety studies. Firstly, SR\'s electrophysiological properties and antiarrhythmic potentials were recorded and assessed at the cell and tissue levels by comprehensively integrating the patch clamp with the Electrical and Optical Mapping systems. Subsequently, the antiarrhythmic effects of SR were validated by aconitine and ouabain-induced arrhythmia in vivo. Finally, the safety of SR as an antiarrhythmic candidate compound was evaluated based on the guidelines of the Comprehensive in Vitro Proarrhythmia Assay (CiPA).
METHODS: The antiarrhythmic effect of SR was evaluated at the in vitro, ex vivo, and in vivo levels.
METHODS: Isolated primary cardiomyocytes and stable cell lines were prepared to explore the electrophysiologic properties of being a multiple ion-channel blocker in vitro by whole-cell patch clamp. Using electrical and optical mapping, the negative chronotropic effect of SR was determined in langendorff-perfused rat or guinea-pig hearts.The antiarrhythmic activity of SR was assessed by the ex vivo
tachyarrhythmia models induced by left coronary artery ligation (LCAL) and isoproterenol (ISO). Canonical models of aconitine and ouabain-induced arrhythmia were used to verify the antiarrhythmic effects in vivo. Finally, the pro-arrhythmic risk of SR was detected in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hSCCMs) using a Microelectrode array (MEA).
RESULTS: Single-cell patch assay validated the multiple ion-channel blockers of SR in transient outward current potassium currents (Ito), l-type calcium currents (ICa-l), and rapid activation delayed rectifier potassium currents (IKr). SR ex vivo depressed heart rates (HR) and ventricular conduction velocity (CV) and prolonged Q-T intervals in a concentration-dependent manner. Consistent with the changes in HRs, SR extended the active time of hearts and increased the action potential duration measured at 90% repolarization (APD90). SR could also significantly lengthen the onset time and curtail the duration of spontaneous ventricular tachycardia (VT) in the ex vivo arrhythmic model induced by LCAL. Meanwhile, SR could also significantly upregulate the programmed electrical stimulation (PES) frequency after the ISO challenge in forming electrical alternans and re-entrant excitation. Furthermore, SR exerted antiarrhythmic effects in the
tachyarrhythmia models induced by aconitine and ouabain in vivo. Notably, the pro-arrhythmic risk of SR was shallow for a moderate inhibition of the human ether-à-go-go-related gene (hERG) channel. Moreover, SR prolonged field potential duration (FPDc) of hSCCMs in a concentration-dependent manner without early after depolarization (EAD) and arrhythmia occurrence.
CONCLUSIONS: Our results indicated that SR manifested as a multiple ion-channel blocker in the electrophysiological properties and exerts antiarrhythmic effects ex vivo and in vivo. Meanwhile, due to the low pro-arrhythmic risk in the hERG inhibition assay and the induction of EAD, SR has great potential as a leading candidate in the treatment of ventricular
tachyarrhythmia.