Excessive ROS production and enhanced autophagy contribute to myocardial injury induced by branched-chain amino acids: Roles for the AMPK-ULK1 signaling pathway and α7nAChR
Excessive ROS production and enhanced autophagy contribute to myocardial injury induced by branched-chain amino acids: Roles for the AMPK-ULK1 signaling pathway and α7nAChR
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过多的 ROS 产生和增强的自噬导致支链氨基酸诱导的心肌损伤：AMPK-ULK1 信号通路和 α7nAChR 的作用
DOI：
10.1016/j.bbadis.2020.165980
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发表时间：
2021-01-01
影响因子：
6.2
通讯作者：
Shen, Fu-Ming
中科院分区：
文献类型：
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作者：
Jiang, Yu-Jie;Sun, Si-Jia;Shen, Fu-Ming
背景和目的：亮氨酸、异亮氨酸和缬氨酸是膳食来源的必需氨基酸，被称为支链氨基酸（BCAA）。支链氨基酸被广泛用作膳食补充剂，以促进肌肉生长和提高运动表现。然而，BCAA对心肌功能的影响在很大程度上是未知的。本研究的目的是调查是否支链氨基酸影响心脏功能，如果是这样的话，进一步探讨潜在的分子基础观察effects.Methods和结果：C57 BL/6 J小鼠随机分为两组，对照组接受溶剂（水）和支链氨基酸组接受2%的支链氨基酸溶于水，连续12周。与对照组相比，BCAA处理显著增加了饮水量，而不改变体重或饮食消耗;心脏组织BCAA水平增加，心脏组织中代表心肌损伤的标志物包括C反应蛋白和心肌肌钙蛋白增加;血清中肌酸激酶、肌酸激酶MB和乳酸脱氢酶增加; Masson染色显示心肌纤维化程度加重，并伴有心肌组织活性氧（ROS）生成增加和超氧化物歧化酶（SOD）活性降低;通过Western印迹和免疫荧光通过LC 3的存在判断，p-AMPK和p-ULK 1均显著增加，自噬也显著增加，在BCAA组中通过透射电子显微镜发现自噬体的数量增加。20 mmol/L BCAA可显著降低H9 C2细胞的存活率，增加细胞内ROS的产生，增加p-AMPK/AMPK和p-ULK 1/ULK 1的表达。用ROS清除剂N-乙酰基-L-半胱氨酸（NAC）处理提高了细胞活力并逆转了ROS变化。20 mmol/L BCAA诱导的H9 C2细胞活力下降可通过阻断AMPK或抑制ULK 1来逆转。此外，阻断AMPK显著降低p-ULK 1/ULK 1，而抑制ULK 1逆转BCAA诱导的LC 3-II/LC 3-I的表达增强。在原代培养的小鼠心肌细胞中进一步证实了BCAA诱导的过量ROS产生和细胞活力降低。PNU-282987对α 7 nAChR的药理学激活可减弱原代鼠心肌细胞中BCA诱导的损伤。然而，该化合物未能抑制BCAA激活AMPK和自噬（LC 3-II/I ratio）。结论：这些结果首次证明BCAA处理小鼠通过触发过量ROS产生和增强AMPK-ULK 1通路依赖的自噬而引起心肌损伤。这些结果表明，抑制ROS的产生或自噬可以减轻BCAA诱导的心肌损伤。
Backgrounds and aims: Leucine, isoleucine, and valine are diet derived and essential amino acids that are termed branched-chain amino acids (BCAA). BCAA are widely used as dietary supplements to boost muscle growth and enhance exercise performance. However, the effects of BCAA on myocardial function are largely unknown. This study was designed to investigate whether BCAA affect heart function and, if so, to further explore the underlying molecular basis for the observed effects.Methods and results: C57BL/6J mice were randomly divided into two groups, the control group received solvent (water) and the BCAA group received 2% BCAA dissolved in water, for a successive period of 12 weeks. Compared with control, BCAA treatment significantly increased water consumption without changing body weight or diet consumption; heart tissue BCAA levels were increased, markers representative of myocardial injury in heart tissue including c-reactive protein and cardiac muscle troponin were increased ; and creatine kinase, creatine kinase-MB, and lactate dehydrogenase were increased in serum; severe myocardial fibrosis was observed by Masson staining, which was accompanied by increased reactive oxygen species (ROS) production and decreased superoxide dismutase activity in heart tissue; both p-AMPK and p-ULK1 were significantly increased as was autophagy, judged by the presence of LC3 by western blotting and immunofluorescence, increased numbers of autophagosomes were found by transmission electron microscopy in the BCAA group. In vitro, 20 mmol/L BCAA significantly decreased cell viability and increased the production of ROS, as well as the expression of p-AMPK/AMPK and p-ULK1/ULK1 in cultured H9C2 cells. Treatment with the ROS scavenger N-acetyl-L-cysteine (NAC) improved cell viability and reversed ROS changes. Decreased H9C2 cell viability induced with 20 mmol/L BCAA was reversed by either blocking AMPK or inhibition of ULK1. Furthermore, blocking AMPK significantly decreased p-ULK1/ULK1, while inhibition of ULK1 reversed the enhanced expression of LC3-II/LC3-I induced by BCAA. Excessive ROS production and decreased cell viability induced by BCAA were further confirmed in primary cultured murine cardiomyocytes. Pharmacological activation of alpha 7nAChR with PNU-282987 attenuated BCAA-induced injury in primary murine cardiomyocytes. However, this compound failed to suppress BCAA activation of AMPK and autophagy (LC3-II/I ratio).Conclusion: These results provide the first evidence that treatment of mice with BCAA induced myocardial injury by triggering excessive ROS production and by enhancing AMPK-ULK1 pathway-dependent autophagy. These findings suggested that inhibition of either ROS production or autophagy may alleviate myocardial injury induced by BCAA.