MCU在低氧诱导肉鸡心肌细胞线粒体损伤中的作用

doi:10.11843/j.issn.0366-6964.2021.012.030

摘要/Abstract

摘要： 旨在探讨线粒体钙单向转运体（mitochondrial calcium uniporter，MCU）介导线粒体Ca²⁺转运是否参与低氧诱导的肉鸡心肌细胞线粒体损伤。试验通过分离白羽肉鸡鸡胚原代心肌细胞，在低氧条件下（3% O₂，5% CO₂，92% N₂）培养24、48、72 h，同时使用RU360抑制MCU的表达并在低氧条件下处理72 h后，使用流式细胞术检测细胞内Ca²⁺浓度、线粒体Ca²⁺浓度、线粒体活性氧水平和线粒体膜电位，检测MCU及其调节因子的mRNA和蛋白表达。结果显示，通过差速贴壁方法培养的心肌细胞纯度可达90%以上；低氧培养24 h，诱导心肌细胞MCU、MICU1 mRNA表达增加（P<0.05），胞浆和线粒体内Ca²⁺浓度显著上升（P<0.05），线粒体膜电位增加（P<0.05）；低氧培养48 h，诱导MCUR1和MICU1 mRNA表达减少（P<0.05），细胞Ca²⁺浓度上升（P<0.05）；低氧培养72 h，诱导MCU mRNA表达增加（P<0.01），细胞和线粒体内Ca²⁺增加（P<0.01），线粒体膜电位下降（P<0.01），活性氧增加（P<0.01）。低氧处理72 h后，与低氧组相比，RU360预处理组细胞和线粒体内Ca²⁺减少，线粒体膜电位上升，活性氧生成减少（P<0.01），MCU mRNA表达减少（P<0.01）。结果表明：低氧诱导MCU上调导致线粒体钙超载，促使线粒体功能降低并发生损伤，进而心肌细胞发生损伤；抑制MCU表达可以减轻低氧诱导的心肌细胞线粒体钙超载，保护线粒体。

关键词: 钙稳态, 低氧, MCU, 心肌细胞

Abstract: The aim of this study was to investigate whether mitochondrial calcium uniporter(MCU) mediated mitochondrial Ca²⁺ transport was involved in hypoxia-induced mitochondrial damage in broiler cardiomyocytes. In this experiment, the primary cardiomyocytes of chicken embryos of white feathered broilers were isolated and cultured for 24, 48 and 72 h under hypoxic conditions (3%O₂, 5%CO₂, 92%N₂). In order to further determine the role of MCU in mitochondrial injury induced by hypoxia, RU360 was pretreated in the hypoxia group for 72 h to inhibit the expression of MCU. The intracellular Ca²⁺ concentration, mitochondrial Ca²⁺ concentration, mitochondrial reactive oxygen species level and mitochondrial membrane potential were detected by flow cytometry. Gene and protein expressions of MCU and its regulatory factors were detected. The results showed that the purity of cardiomyocytes cultured by differential velocity adherent method could reach over 90%. After 24 h of hypoxic culture, the expression of MCU and MICU1 genes were increased (P<0.05), the concentrations of Ca²⁺ in cytoplasm and mitochondria were significantly increased (P<0.05), and the mitochondrial membrane potential was increased (P<0.05). The expression levels of MCUR1 and MICU1 mRNA were decreased (P<0.05) and intracellular Ca²⁺ concentration was increased (P<0.05) after 48 h hypoxia culture. After 72 h of hypoxic culture, MCU mRNA expression, intracellular and mitochondrial Ca²⁺ were increased (P<0.01), and reactive oxygen species were increased (P<0.01), while mitochondial membrane potential decreased (P<0.01). Compared with the 72 h hypoxic group, the Ca²⁺ in cells and mitochondria decreased, the mitochondrial membrane potential increased and the reactive oxygen species decreased in the RU360 preconditioning group (P<0.01), the expression of MCU mRNA was decreased (P<0.01). The results showed that hypoxia-induced MCU upregulation leads to mitochondrial calcium overload, causing mitochondrial dysfunction and cardiomyocyte injury. Inhibition of MCU expression can reduce hypoxia-induced mitochondrial calcium overload and protect mitochondrial function.

Key words: calcium steady state, hypoxic, MCU, myocardial cells

中图分类号:

S856.2
Q256

庞聪颖, 乔娜, 陈汉明, 马欣艳, 张辉, 潘家强, 唐兆新, 李英. MCU在低氧诱导肉鸡心肌细胞线粒体损伤中的作用[J]. 畜牧兽医学报, 2021, 52(12): 3641-3650.

PANG Congying, QIAO Na, CHEN Hanming, MA Xinyan, ZHANG Hui, PAN Jiaqiang, TANG Zhaoxin, LI Ying. Role of MCU in Mitochondrial Damage Induced by Hypoxia in Broiler Cardiomyocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3641-3650.

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