DataSheet2_Dexmedetomidine Protects Human Cardiomyocytes Against Ischemia-Reperfusion Injury Through α2-Adrenergic Receptor/AMPK-Dependent Autophagy.docx

Background: Ischemia-reperfusion injury (I/R) strongly affects the prognosis of children with complicated congenital heart diseases (CHDs) who undergo long-term cardiac surgical processes. Recently, the α2-adrenergic receptor agonist Dexmedetomidine (Dex) has been reported to protect cardiomyocytes (CMs) from I/R in cellular models and adult rodent models. However, whether and how Dex may protect human CMs in young children remains largely unknown.
Methods and Results: Human ventricular tissue from tetralogy of Fallot (TOF) patients and CMs derived from human-induced pluripotent stem cells (iPSC-CMs) were used to assess whether and how Dex protects human CMs from I/R. The results showed that when pretreated with Dex, the apoptosis marker-TUNEL and cleaved caspase 3 in the ventricular tissue were significantly reduced. In addition, the autophagy marker LC3II was significantly increased compared with that of the control group. When exposed to the hypoxia/reoxygenation process, iPSC-CMs pretreated with Dex also showed reduced TUNEL and cleaved caspase 3 and increased LC3II. When the autophagy inhibitor (3-methyladenine, 3-MA) was applied to the iPSC-CMs, the protective effect of Dex on the CMs was largely blocked. In addition, when the fusion of autophagosomes with lysosomes was blocked by Bafilomycin A1, the degradation of p62 induced by Dex during the autophagy process was suspended. Moreover, when pretreated with Dex, both the human ventricle and the iPSC-CMs expressed more AMP-activated protein kinase (AMPK) and phospho AMPK (pAMPK) during the I/R process. After AMPK knockout or the use of an α2-adrenergic receptor antagonist-yohimbine, the protection of Dex and its enhancement of autophagy were inhibited.
Conclusion: Dex protects young human CMs from I/R injury, and α2-adrenergic receptor/AMPK-dependent autophagy plays an important role during this process. Dex may have a therapeutic effect for children with CHD who undergo long-term cardiac surgical processes.

研究背景：缺血再灌注损伤（Ischemia-reperfusion injury, I/R）会显著影响接受长期心脏外科手术的复杂性先天性心脏病（complicated congenital heart diseases, CHDs）患儿的预后。近期有研究表明，α2肾上腺素能受体激动剂右美托咪定（Dexmedetomidine, Dex）可在细胞模型及成年啮齿类动物模型中保护心肌细胞（cardiomyocytes, CMs）免受缺血再灌注损伤。然而，右美托咪定能否以及如何保护幼儿的人类心肌细胞，目前仍尚不明确。

研究方法与结果：本研究采用法洛四联症（tetralogy of Fallot, TOF）患者的人类心室组织，以及人类诱导多能干细胞来源心肌细胞（human-induced pluripotent stem cell-derived cardiomyocytes, iPSC-CMs），以探究右美托咪定能否以及如何保护人类心肌细胞免受缺血再灌注损伤。结果显示，经右美托咪定预处理后，心室组织中的细胞凋亡标志物TUNEL及活化半胱氨酸天冬氨酸蛋白酶3（cleaved caspase 3）水平显著降低。此外，与对照组相比，自噬标志物LC3II的表达量显著升高。在暴露于缺氧/复氧（hypoxia/reoxygenation）环境时，经右美托咪定预处理的iPSC-CMs同样表现出TUNEL及cleaved caspase 3水平降低、LC3II表达升高的现象。当向iPSC-CMs中加入自噬抑制剂3-甲基腺嘌呤（3-methyladenine, 3-MA）后，右美托咪定对心肌细胞的保护作用被显著阻断。此外，当巴弗洛霉素A1（Bafilomycin A1）阻断自噬体与溶酶体的融合时，右美托咪定在自噬过程中诱导的p62蛋白降解被抑制。进一步研究发现，经右美托咪定预处理后，无论是人类心室组织还是iPSC-CMs，在缺血再灌注过程中腺苷酸活化蛋白激酶（AMP-activated protein kinase, AMPK）及磷酸化AMPK（phospho AMPK, pAMPK）的表达均有所升高。当AMPK基因敲除或使用α2肾上腺素能受体拮抗剂育亨宾（yohimbine）后，右美托咪定的保护作用及其对自噬的增强效应均受到抑制。

研究结论：右美托咪定可保护幼儿人类心肌细胞免受缺血再灌注损伤，且α2肾上腺素能受体/AMPK依赖的自噬过程在其中发挥了重要作用。右美托咪定或可用于接受长期心脏外科手术的先天性心脏病患儿的治疗。