Doctoral Program in Sports Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan.
Laboratory of Clinical Examination/Sports Medicine, Department of Clinical Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
Biochem Biophys Res Commun. 2022 Jun 11;608:59-65. doi: 10.1016/j.bbrc.2022.03.093. Epub 2022 Mar 19.
Cryotherapy is one of the most common treatments for trauma or fatigue in the field of sports medicine. However, the molecular biological effects of acute cold exposure on skeletal muscle remain unclear. Therefore, we used zebrafish, which have recently been utilized as an animal model for skeletal muscle, to comprehensively investigate and selectively clarify the time-course changes induced by cryotherapy. Zebrafish were exposed intermittently to cold stimulation three times for 15 min each. Thereafter, skeletal muscle samples were collected after 15 min and 1, 2, 4, and 6 h. mRNA sequencing revealed the involvement of trim63a, fbxo32, fbxo30a, and klhl38b in "protein ubiquitination" from the top 10 most upregulated genes. Subsequently, we examined the time-course changes of the four genes by quantitative PCR, and their expression peaked 2 h after cryotherapy and returned to baseline after 6 h. Moreover, the proteins encoded by trim63a and fbxo32 (muscle-specific RING finger protein 1 [MuRF1] and muscle atrophy F-box, respectively), which are known to be major genes encoding E3 ubiquitin ligases, were examined by western blotting, and MuRF1 expression displayed similar temporal changes as trim63a expression. These findings suggest that acute cold exposure transiently upregulates E3 ubiquitin ligases, especially MuRF1; thus, cryotherapy may contribute to the treatment of trauma or fatigue by promoting protein processing.
冷冻疗法是运动医学领域中治疗创伤或疲劳最常用的方法之一。然而，急性冷暴露对骨骼肌的分子生物学影响尚不清楚。因此，我们使用斑马鱼作为骨骼肌的动物模型，来全面研究和选择性阐明冷冻疗法诱导的时间过程变化。斑马鱼被间歇性地暴露于冷刺激三次，每次 15 分钟。此后，在 15 分钟、1 小时、2 小时、4 小时和 6 小时后收集骨骼肌样本。mRNA 测序显示，在 top10 上调基因中，trim63a、fbxo32、fbxo30a 和 klhl38b 参与“蛋白质泛素化”。随后，我们通过定量 PCR 检查了这四个基因的时间过程变化，它们的表达在冷冻治疗后 2 小时达到峰值，并在 6 小时后恢复到基线。此外，trim63a 和 fbxo32 编码的蛋白质（肌肉特异性环指蛋白 1 [MuRF1]和肌肉萎缩 F-box），这两种蛋白已知是主要的 E3 泛素连接酶编码基因，通过 Western blot 进行了检查，MuRF1 的表达与 trim63a 的表达呈现相似的时间变化。这些发现表明，急性冷暴露短暂地上调 E3 泛素连接酶，特别是 MuRF1；因此，冷冻疗法可能通过促进蛋白质处理来治疗创伤或疲劳。
