Calenduloside E suppresses calcium overload by promoting the interaction between L-type calcium channels and Bcl2-associated athanogene 3 to alleviate myocardial ischemia/reperfusion injury,Journal of Advanced Research

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Calenduloside E suppresses calcium overload by promoting the interaction between L-type calcium channels and Bcl2-associated athanogene 3 to alleviate myocardial ischemia/reperfusion injury
Journal of Advanced Research ( IF 11.4 ) Pub Date : 2020-10-31 , DOI: 10.1016/j.jare.2020.10.005
Ruiying Wang _{1,

2,

3} , Min Wang _{1,

2,

3} , Jiahui Zhou _{1,

2,

3} , Ziru Dai _{1,

2,

3} , Guibo Sun _{1,

2,

3} , Xiaobo Sun _{1,

2,

3}

Affiliation

Introduction

Intracellular calcium overload is an important contributor to myocardial ischemia/reperfusion (MI/R) injury. Total saponins of the traditional Chinese medicinal plant Aralia elata (Miq.) Seem. (AS) are beneficial for treating MI/R injury, and Calenduloside E (CE) is the main active ingredient of AS.

Objectives

This study aimed to investigate the effects of CE on MI/R injury and determine its specific regulatory mechanisms.

Methods

To verify whether CE mediated cardiac protection in vivo and in vitro, we performed MI/R surgery in SD rats and subjected neonatal rat ventricular myocytes (NRVMs) to hypoxia-reoxygenation (HR). CE’s cardioprotective against MI/R injury was detected by Evans blue/TTC staining, echocardiography, HE staining, myocardial enzyme levels. Impedance and field potential recording, and patch-clamp techniques of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to detect the function of L-type calcium channels (LTCC). The mechanisms underlying between CE and LTCC was studied through western blot, immunofluorescence, and immunohistochemistry. Drug affinity responsive target stability (DARTS) and co-immunoprecipitation (co-IP) used to further clarify the effect of CE on LTCC and BAG3.

Results

We found that CE protected against MI/R injury by inhibiting calcium overload. Furthermore, CE improved contraction and field potential signals of hiPSC-CMs and restored sarcomere contraction and calcium transient of adult rat ventricular myocytes (ARVMs). Moreover, patch-clamp data showed that CE suppressed increased L-type calcium current (I_Ca,L) caused by LTCC agonist, proving that CE could regulate calcium homeostasis through LTCC. Importantly, we found that CE promoted the interaction between LTCC and Bcl2-associated athanogene 3 (BAG3) by co-IP and DARTS.

Conclusion

Our results demonstrate that CE enhanced LTCC-BAG3 interaction to reduce MI/R induced-calcium overload, exerting a cardioprotective effect.

中文翻译：

金盏花苷 E 通过促进 L 型钙通道与 Bcl2 相关的 athanogene 3 之间的相互作用抑制钙超载以减轻心肌缺血/再灌注损伤

介绍

细胞内钙超载是导致心肌缺血/再灌注 (MI/R) 损伤的重要因素。中药材Aralia elata (Miq.) Seem的总皂苷。(AS) 有利于治疗 MI/R 损伤，而金盏花苷 E (CE) 是 AS 的主要活性成分。

目标

本研究旨在调查 CE 对 MI/R 损伤的影响并确定其特定的调节机制。

方法

为了验证 CE 是否在体内和体外介导心脏保护，我们对 SD 大鼠进行了 MI/R 手术，并对新生大鼠心室肌细胞 (NRVM) 进行了缺氧复氧 (HR)。通过埃文斯蓝/TTC染色、超声心动图、HE染色、心肌酶水平检测CE对MI/R损伤的心脏保护作用。使用人诱导多能干细胞衍生心肌细胞 (hiPSC-CMs) 的阻抗和场电位记录以及膜片钳技术检测 L 型钙通道 (LTCC) 的功能。通过蛋白质印迹、免疫荧光和免疫组织化学研究了 CE 和 LTCC 之间的潜在机制。药物亲和力反应靶稳定性 (DARTS) 和免疫共沉淀 (co-IP) 用于进一步阐明 CE 对 LTCC 和 BAG3 的影响。

结果

我们发现 CE 通过抑制钙超载来防止 MI/R 损伤。此外，CE 改善了 hiPSC-CM 的收缩和场电位信号，并恢复了成年大鼠心室肌细胞 (ARVM) 的肌节收缩和钙瞬变。此外，膜片钳数据显示 CE 抑制了由 LTCC 激动剂引起的 L 型钙电流（I _Ca,L）增加，证明 CE 可以通过 LTCC 调节钙稳态。重要的是，我们发现 CE 通过 co-IP 和 DARTS 促进了 LTCC 和 Bcl2 相关的 athanogene 3 (BAG3) 之间的相互作用。