Myology Laboratory, Institute of Biomedical Problems RAS, 123007 Moscow, Russia.
Int J Mol Sci. 2022 Mar 2;23(5):2751. doi: 10.3390/ijms23052751.
It is well-established that prolonged exposure to real or simulated microgravity/disuse conditions results in a significant reduction in the rate of muscle protein synthesis (PS) and loss of muscle mass. Muscle protein synthesis is largely dependent upon translational capacity (ribosome content), the regulation of which is poorly explored under conditions of mechanical unloading. Glycogen synthase kinase-3 (GSK-3) (a negative regulator of PS) is known to be activated in rat soleus muscle under unloading conditions. We hypothesized that inhibition of GSK-3 activity under disuse conditions (hindlimb suspension, HS) would reduce disuse-induced downregulation of ribosome biogenesis in rat soleus muscle. Wistar rats were randomly divided into four groups: (1) vivarium control (C), (2) vivarium control + daily injections (4 mg/kg) of AR-A014418 (GSK-3 inhibitor) for 7 days, (3) 7-day HS, (4) 7-day HS + daily injections (4 mg/kg) of AR-A014418. GSK-3beta and glycogen synthase 1 (GS-1) phosphorylation levels were measured by Western-blotting. The key markers of ribosome biogenesis were assessed via agarose gel-electrophoresis and RT-PCR. The rate of muscle PS was assessed by puromycin-based SUnSET method. As expected, 7-day HS resulted in a significant decrease in the inhibitory Ser9 GSK-3beta phosphorylation and an increase in GS-1 (Ser641) phosphorylation compared to the C group. Treatment of rats with GSK-3 inhibitor prevented HS-induced increase in GS1 (Ser641) phosphorylation, which was indicative of GSK-3 inhibition. Administration of GSK-3 inhibitor partly attenuated disuse-induced downregulation of c-Myc expression as well as decreases in the levels of 45S pre-rRNA and 18S + 28S rRNAs. These AR-A014418-induced alterations in the markers of ribosome biogenesis were paralleled with partial prevention of a decrease in the rate of muscle PS. Thus, inhibition of GSK-3 during 7-day HS is able to partially attenuate the reductions in translational capacity and the rate of PS in rat soleus muscle.
众所周知，长时间暴露在模拟或真实的微重力/停用状态下会导致肌肉蛋白质合成（PS）的速率显著降低，肌肉质量也会随之减少。肌肉蛋白质合成在很大程度上依赖于翻译能力（核糖体含量），而在机械卸载的情况下，其调节机制还未被充分探索。糖原合成酶激酶-3（GSK-3）（PS 的负调节剂）在卸载条件下的大鼠比目鱼肌中被证实被激活。我们假设在停用状态下（后肢悬停，HS）抑制 GSK-3 活性会减少大鼠比目鱼肌中核糖体生物发生的停用诱导下调。Wistar 大鼠被随机分为四组：（1）饲养室对照（C），（2）饲养室对照+每天注射（4mg/kg）AR-A014418（GSK-3 抑制剂）持续 7 天，（3）7 天 HS，（4）7 天 HS+每天注射（4mg/kg）AR-A014418。通过 Western-blotting 测定 GSK-3β和糖原合酶 1（GS-1）磷酸化水平。通过琼脂糖凝胶电泳和 RT-PCR 评估核糖体生物发生的关键标志物。通过基于嘌呤霉素的 SUnSET 方法评估肌肉 PS 率。正如预期的那样，与 C 组相比，7 天 HS 导致抑制性 Ser9 GSK-3β磷酸化显著降低，GS-1（Ser641）磷酸化增加。用 GSK-3 抑制剂处理大鼠可防止 HS 诱导的 GS1（Ser641）磷酸化增加，这表明 GSK-3 被抑制。GSK-3 抑制剂的给药部分减轻了停用引起的 c-Myc 表达下调以及 45S 前 rRNA 和 18S+28S rRNA 水平降低。AR-A014418 诱导的核糖体生物发生标志物的这些变化与肌肉 PS 率降低部分得到预防相平行。因此，在 7 天 HS 期间抑制 GSK-3 能够部分减轻大鼠比目鱼肌中翻译能力和 PS 率的降低。
