School of Nursing, Hirosaki Gakuin University, Hirosaki, Aomori, Japan.
Graduate School of Health and Sports Science, Juntendo University, Inzai, Chiba, Japan.
PLoS One. 2020 Dec 9;15(12):e0243660. doi: 10.1371/journal.pone.0243660. eCollection 2020.
Excessive nitric oxide (NO) production and mitochondrial dysfunction can activate protein degradation in disuse-induced skeletal muscle atrophy. However, the increase in NO production in atrophied muscles remains controversial. In addition, although several studies have investigated the PTEN-induced kinase 1 (PINK1)/Parkin pathway, a mitophagy pathway, in atrophied muscle, the involvement of this pathway in soleus muscle atrophy is unclear. In this study, we investigated the involvement of neuronal nitric oxide synthase (nNOS) and the PINK1/Parkin pathway in soleus muscle atrophy induced by 14 days of hindlimb unloading (HU) in adult rats. HU lowered the weight of the soleus muscles. nNOS expression showed an increase in atrophied soleus muscles. Although HU increased malondialdehyde as oxidative modification of the protein, it decreased 6-nitrotryptophan, a marker of protein nitration. Additionally, the nitrosocysteine content and S-nitrosylated Parkin were not altered, suggesting the absence of excessive nitrosative stress after HU. The expression of PINK1 and Parkin was also unchanged, whereas the expression of heat shock protein 70 (HSP70), which is required for Parkin activity, was reduced in atrophied soleus muscles. Moreover, we observed accumulation and reduced ubiquitination of high molecular weight mitofusin 2, which is a target of Parkin, in atrophied soleus muscles. These results indicate that excessive NO is not produced in atrophied soleus muscles despite nNOS accumulation, suggesting that excessive NO dose not mediate in soleus muscle atrophy at least after 14 days of HU. Furthermore, the PINK1/Parkin pathway may not play a role in mitophagy at this time point. In contrast, the activity of Parkin may be downregulated because of reduced HSP70 expression, which may contribute to attenuated degradation of target proteins in the atrophied soleus muscles after 14 days of HU. The present study provides new insights into the roles of nNOS and a protein degradation pathway in soleus muscle atrophy.
过量的一氧化氮（NO）产生和线粒体功能障碍可激活废用性肌萎缩中的蛋白质降解。然而，萎缩肌肉中 NO 产生的增加仍存在争议。此外，尽管有几项研究已经研究了在萎缩肌肉中的 PTEN 诱导的激酶 1（PINK1）/Parkin 途径（一种自噬途径），但其在比目鱼肌萎缩中的参与尚不清楚。在这项研究中，我们研究了神经元型一氧化氮合酶（nNOS）和 PINK1/Parkin 途径在成年大鼠 14 天的后肢去负荷（HU）引起的比目鱼肌萎缩中的作用。HU 降低了比目鱼肌的重量。在萎缩的比目鱼肌中，nNOS 表达增加。尽管 HU 增加了丙二醛作为蛋白质的氧化修饰，但它降低了 6-硝基色氨酸，这是蛋白质硝化的标志物。此外，亚硝酰半胱氨酸含量和 S-亚硝基化 Parkin 没有改变，表明 HU 后没有过度的硝化应激。PINK1 和 Parkin 的表达也没有改变，而活性所需的热休克蛋白 70（HSP70）的表达在萎缩的比目鱼肌中降低。此外，我们观察到在萎缩的比目鱼肌中积累和减少了高分子量融合蛋白 2 的泛素化，融合蛋白 2 是 Parkin 的靶标。这些结果表明，尽管 nNOS 积累，但在萎缩的比目鱼肌中没有产生过量的 NO，这表明至少在 14 天后的 HU 中，过量的 NO 不会介导比目鱼肌萎缩。此外，在此时点，PINK1/Parkin 途径可能不会在自噬中起作用。相反，由于 HSP70 表达降低，Parkin 的活性可能被下调，这可能导致 HU 后 14 天萎缩的比目鱼肌中靶蛋白的降解减弱。本研究为 nNOS 和蛋白质降解途径在比目鱼肌萎缩中的作用提供了新的见解。
