Purpose: Autophagy caused by ischemia/reperfusion (I/R) increases the extent of cardiomyocyte damage. Melatonin (Mel) diminishes cardiac injury through regulating autophagy and mitochondrial dynamics. However, illustrating the specific role of mitophagy in the cardioprotective effects of melatonin remains a challenge. The aim of our research was to investigate the impact and underlying mechanisms of melatonin in connection with mitophagy during anoxia/reoxygenation (A/R) injury in H9c2 cells.
Methods: H9c2 cells were pretreated with melatonin with or without the melatonin membrane receptor 2 (MT2) antagonist 4-P-PDOT, the MT2 agonist IIK7 and the sirtuin 3 (SIRT3) inhibitor 3-TYP for 4 hours and then subjected to A/R injury. Cell viability, cellular apoptosis, necrosis levels and oxidative markers were assessed. The expression of SIRT3 and forkhead box O3a (FoxO3a), mitochondrial function and the levels of mitophagy-related proteins were also evaluated.
Results: A/R injury provoked enhanced mitophagy in H9c2 myocytes. In addition, increased mitophagy was correlated with decreased cellular viability, increased oxidative stress and mitochondrial dysfunction in H9c2 cells. However, melatonin pretreatment notably increased cell survival and decreased cell apoptosis and oxidative response after A/R injury, accompanied by restored mitochondrial function. The inhibition of excessive mitophagy is involved in the cardioprotective effects of melatonin, as shown by the decreased expression of the mitophagy-related molecules Parkin, Beclin1, and BCL2-interacting protein 3-like (BNIP3L, best known as NIX) and decreased light chain 3 II/light chain 3 I (LC3 II/LC3 I) ratio and upregulation of p62 expression. Moreover, the decreased expression of SIRT3 and FoxO3a in A/R-injured H9c2 cells was abrogated by melatonin, but these beneficial effects were attenuated by the MT2 antagonist 4-P-PDOT or the SIRT3 inhibitor 3-TYP and enhanced by the MT2 agonist IIK7.
Conclusion: These results indicate that melatonin protects H9c2 cells during A/R injury through suppressing excessive mitophagy by activating the MT2/SIRT3/FoxO3a pathway. Melatonin may be a useful candidate for alleviating myocardial ischemia/reperfusion (MI/R) injury in the future, and the MT2 receptor might become a therapeutic target.

Keywords: melatonin, mitophagy, anoxia/reoxygenation injury, melatonin receptor, SIRT3




中文翻译：

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褪黑激素通过抑制 H9c2 细胞中的 MT2/SIRT3/FoxO3a 信号通路过度线粒体自噬来减轻缺氧/复氧损伤。

目的：缺血/再灌注（I/R）引起的自噬会增加心肌细胞损伤的程度。褪黑激素 (Mel) 通过调节自噬和线粒体动力学来减轻心脏损伤。然而，阐明线粒体自噬在褪黑激素的心脏保护作用中的具体作用仍然是一个挑战。我们研究的目的是研究褪黑激素与 H9c2 细胞缺氧/复氧 (A/R) 损伤过程中线粒体自噬相关的影响和潜在机制。
方法：H9c2 细胞用褪黑激素与或不与褪黑激素膜受体 2 (MT2) 拮抗剂 4-P-PDOT、MT2 激动剂 IIK7 和 sirtuin 3 (SIRT3) 抑制剂 3-TYP 预处理 4 小时，然后进行 A/R 损伤. 评估细胞活力、细胞凋亡、坏死水平和氧化标志物。还评估了 SIRT3 和叉头盒 O3a (FoxO3a) 的表达、线粒体功能和线粒体自噬相关蛋白的水平。
结果：A/R 损伤引起 H9c2 肌细胞线粒体自噬增强。此外，线粒体自噬增加与 H9c2 细胞中细胞活力降低、氧化应激增加和线粒体功能障碍相关。然而，褪黑激素预处理显着增加了细胞存活率，减少了 A/R 损伤后的细胞凋亡和氧化反应，伴随着线粒体功能的恢复。过度线粒体自噬的抑制与褪黑激素的心脏保护作用有关，如线粒体自噬相关分子 Parkin、Beclin1 和 BCL2 相互作用蛋白 3 样（BNIP3L，最著名的是 NIX）的表达减少和轻链减少所示3 II/轻链 3 I (LC3 II/LC3 I) 比率和 p62 表达的上调。此外，褪黑激素消除了 A/R 损伤的 H9c2 细胞中 SIRT3 和 FoxO3a 的表达降低，
结论：这些结果表明，褪黑激素通过激活 MT2/SIRT3/FoxO3a 通路抑制过度线粒体自噬，从而在 A/R 损伤期间保护 H9c2 细胞。褪黑激素可能是未来缓解心肌缺血/再灌注 (MI/R) 损伤的有用候选药物，而 MT2 受体可能成为治疗靶点。

关键词：褪黑激素，线粒体自噬，缺氧/复氧损伤，褪黑激素受体，SIRT3