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 α7nAChR 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.

中文翻译：

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过量的ROS产生和自噬增强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

背景和目的

亮氨酸，异亮氨酸和缬氨酸是饮食来源的必需氨基酸，被称为支链氨基酸（BCAA）。BCAA被广泛用作膳食补充剂，以促进肌肉生长和增强运动表现。但是，BCAA对心肌功能的影响在很大程度上尚不清楚。本研究旨在调查BCAA是否会影响心脏功能，如果是，则进一步探讨观察到的影响的潜在分子基础。

方法与结果

将C57BL / 6J小鼠随机分为两组，对照组接受溶剂（水），BCAA组接受2％溶于水中的BCAA，连续12周。与对照组相比，BCAA处理显着增加了水的消耗，而没有改变体重或饮食的消耗。心脏组织中BCAA水平升高，代表心脏组织中心肌损伤的标志物（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和乳酸脱氢酶升高；通过Masson染色观察到严重的心肌纤维化，并伴有心脏组织中活性氧（ROS）生成增加和超氧化物歧化酶活性降低。p-AMPK和p-ULK1均与自噬显着增加，在体外，20 mmol / L BCAA显着降低了细胞活力并增加了ROS的产生，以及在培养的H9C2细胞中p-AMPK / AMPK和p-ULK1 / ULK1的表达。用ROS清除剂N处理-乙酰基-L-半胱氨酸（NAC）可提高细胞活力并逆转ROS的变化。通过阻断AMPK或抑制ULK1，可逆转20 mmol / L BCAA诱导的H9C2细胞活力降低。此外，阻断AMPK可显着降低p-ULK1 / ULK1，而抑制ULK1可逆转BCAA诱导的LC3-II / LC3-I增强表达。在原代培养的鼠心肌细胞中进一步证实了过量的ROS产生和由BCAA诱导的细胞活力降低。用PNU-282987药理活化α7nAChR可减轻BCAA诱导的原代鼠心肌细胞损伤。但是，该化合物不能抑制AMPK的BCAA活化和自噬（LC3-II / I比）。

结论

这些结果提供了第一个证据，即用BCAA处理小鼠会触发过多的ROS产生并增强AMPK-ULK1途径依赖性自噬，从而引起心肌损伤。这些发现表明，抑制ROS产生或自噬可以减轻由BCAA引起的心肌损伤。