Graduate School of Sports and Health Science, Fukuoka University, Fukuoka, Japan.
The Fukuoka University Institute for Physical Activity, Fukuoka, Japan.
J Physiol. 2019 Nov;597(21):5145-5159. doi: 10.1113/JP277019. Epub 2019 Oct 9.
DNA methylation may play an important role in regulating gene expression in skeletal muscle to adapt to physical activity and inactivity. Neuronal nitric oxide synthase (nNOS) in skeletal muscle is a key regulator of skeletal muscle mass; however, it is unclear whether nNOS expression is regulated by DNA methylation. We found that 1 week of cast immobilization increased nNOS DNA methylation levels and downregulated nNOS gene expression in atrophic slow-twitch soleus muscle from the mouse leg. These changes were not detected in non-atrophic fast-twitch extensor digitorum longus muscle. Twelve hours of cast immobilization decreased nNOS gene expression, whereas nNOS DNA methylation levels were unchanged, suggesting that downregulation of nNOS gene expression by short-term muscle inactivity is independent of the DNA methylation pattern. These findings contribute to a better understanding of the maintenance of skeletal muscle mass and prevention of muscle atrophy by epigenetic mechanisms via the nNOS/NO pathway.
DNA methylation is a mechanism that controls gene expression in skeletal muscle under various environmental stimuli, such as physical activity and inactivity. Neuronal nitric oxide synthase (nNOS) regulates muscle atrophy in skeletal muscle. However, the mechanisms regulating nNOS expression in atrophic muscle remain unclear. We hypothesized that nNOS expression in atrophic muscle is regulated by DNA methylation of the nNOS promotor in soleus (Sol; slow-twitch fibre dominant) and extensor digitorum longus (EDL; fast-twitch fibre dominant) muscles. One week of cast immobilization induced significant muscle atrophy in Sol but not in EDL. We showed that 1 week of cast immobilization increased nNOS DNA methylation levels in Sol, although only a minor change was detected in EDL. Consistent with the increased DNA methylation levels in atrophic Sol, the gene expression levels of total nNOS and nNOSµ (i.e. the major splicing variant of nNOS in skeletal muscle) decreased. The abundance of the nNOS protein and cell membrane (especially type IIa fibre) immunoreactivity also decreased in atrophic Sol. These changes were not observed in EDL after 1 week of cast immobilization. Furthermore, despite the lack of significant atrophy, 12 h of cast immobilization decreased gene expression levels of total nNOS and nNOSµ in Sol. However, no association was detected between nNOS DNA methylation and gene expression. The expression of the nNOSβ gene, another splicing variant of nNOS, in EDL was unchanged by cast immobilization, whereas its expression was not detected in Sol. We concluded that chronic adaptation of nNOS gene expression in cast immobilized muscle may involve nNOS DNA methylation.
DNA 甲基化可能在调节骨骼肌中的基因表达以适应体力活动和不活动方面发挥重要作用。骨骼肌中的神经元型一氧化氮合酶（nNOS）是骨骼肌质量的关键调节剂；然而，nNOS 表达是否受 DNA 甲基化调控尚不清楚。我们发现，1 周的石膏固定使小鼠腿部萎缩的慢收缩比目鱼肌中的 nNOS DNA 甲基化水平增加，并下调了 nNOS 基因表达。在非萎缩的快收缩伸趾长肌中未检测到这些变化。12 小时的石膏固定使 nNOS 基因表达减少，而 nNOS DNA 甲基化水平不变，表明短期肌肉失活引起的 nNOS 基因表达下调与 DNA 甲基化模式无关。这些发现有助于更好地理解通过 nNOS/NO 途径的表观遗传机制来维持骨骼肌质量和预防肌肉萎缩。
DNA 甲基化是一种在各种环境刺激下控制骨骼肌基因表达的机制，例如体力活动和不活动。神经元型一氧化氮合酶（nNOS）调节骨骼肌中的肌肉萎缩。然而，调节萎缩肌肉中 nNOS 表达的机制尚不清楚。我们假设 nNOS 在萎缩肌肉中的表达受 nNOS 启动子的 DNA 甲基化调节在比目鱼肌（Sol；慢收缩纤维占优势）和伸趾长肌（EDL；快收缩纤维占优势）中。1 周的石膏固定使 Sol 明显萎缩，但 EDL 没有萎缩。我们发现，1 周的石膏固定增加了 Sol 中的 nNOS DNA 甲基化水平，而在 EDL 中仅检测到较小的变化。与萎缩 Sol 中的 DNA 甲基化水平增加一致，总 nNOS 和 nNOSµ（即 nNOS 在骨骼肌中的主要剪接变体）的基因表达水平降低。nNOS 蛋白的丰度和细胞膜（特别是 IIa 型纤维）免疫反应性也在萎缩的 Sol 中减少。1 周石膏固定后，EDL 中未观察到这些变化。此外，尽管没有明显的萎缩，12 小时的石膏固定也降低了 Sol 中总 nNOS 和 nNOSµ 的基因表达水平。然而，nNOS DNA 甲基化与基因表达之间没有关联。另一种 nNOS 剪接变体 nNOSβ在 EDL 中的表达不受石膏固定的影响，而在 Sol 中则未检测到。我们得出结论，慢性适应石膏固定肌肉中的 nNOS 基因表达可能涉及 nNOS DNA 甲基化。
