The role of myosin II in tendon repair under glucose control

肌腱损伤和病症对经济成本和满足治疗需求的能力产生严重的负面影响。 患者的职业、娱乐和健康活动。尽管不断努力改善治疗效果， 对于肌腱修复的方式，尚未建立真正有效和高效的治疗方法。我们的长期 目的是了解肌腱有限再生潜力的机制，并开发新的 用于刺激肌腱修复的方法。与胚胎和新生儿肌腱不同， 不具有天然肌腱结构和机械性能。瘢痕愈合肌腱伴异常 力学性能损伤肌腱的结构和功能恢复的关键挑战之一是如何 增强参与肌腱愈合的肌腱祖细胞的修复能力，包括如何 刺激它们的成腱分化以及如何使它们重建腱基质结构。的结果 临床和转化研究表明葡萄糖代谢和肌腱愈合能力之间存在密切联系。 我们发现，2-脱氧葡萄糖（2DG），一种葡萄糖抑制剂，刺激胶原纤维的恢复 在受伤的肌腱对齐。2DG对肌腱愈合作用的蛋白质组学研究表明， 2DG刺激损伤肌腱中的肌动蛋白细胞骨架信号。但我们发现，中 肌腱实质表达肌型肌球蛋白II组分，但表达强烈下调 当这些分子受损时，而2DG恢复了这些变化。 此外，损伤的肌腱来源的祖细胞对2DG有反应，上调了肌型胶原的表达。 肌球蛋白轻链和重排的肌动蛋白细胞骨架。以前的研究表明，肌动蛋白 胶原纤维排列需要细胞骨架组织和肌球蛋白II活性。总之，我们 假设损伤肌腱中肌型肌球蛋白II分子上调可能刺激胶原纤维 对齐。此外，葡萄糖控制肌肉型肌球蛋白II分子和肌动蛋白的表达。 细胞骨架组织为了验证这些假设，我们提出了两个目标。在目标1中，我们将确定更改 肌球蛋白II分子在肌腱损伤，愈合和再生过程中。肌球蛋白的时空变化 II组分（肌球蛋白重链和轻链）将在使用四只小鼠跟腱的损伤肌腱中定义 肌腱损伤模型。在目标2中，我们将确定肌球蛋白II在损伤肌腱源性 细胞（inTPC）。InTPC将在单一或3D培养物中用2DG处理，并且肌球蛋白表达的变化 II分子，RhoA/Rac活性，胶原纤维排列和机械性能将被检查。此外，本发明还提供了一种方法， 在相同的模型中，将确定肌肉肌球蛋白II分子的功能获得和丧失的影响。 据我们所知，目前还不知道肌肉和非肌肉肌球蛋白II分子是否有一个 肌腱的唯一功能。因此，该项目符合R21的范围，其成果提供了第一个 来回答这个问题。

Tendon injury and disorders provide a severe negative impact on the economic cost and the ability to meet the patients' occupational, recreational, and health activities. Despite continuous efforts to improve the therapeutic modalities for tendon repair, truly effective and efficient therapies have not been established yet. Our long-term goal is to understand the mechanisms underlying the limited regenerative potential of tendons and develop new methods for stimulating tendon repair. Unlike embryonic and neonatal tendons, neo-formed tendons after injury do not possess native tendon structure and mechanical properties. Healing tendons from scar with abnormal mechanical properties. One of the key challenges for recovery of structure and function of injured tendons is how to potentiate the reparative capacity of the tendon progenitor cells involved in tendon healing, including how to stimulate their tenogenic differentiation and how to make them rebuild tendon matrix structure. The results from clinical and translational studies indicate a close link between glucose metabolism and tendon healing capacity. We have found that 2-deoxy glucose (2DG), an inhibitor of glucose, stimulated recovery of collagen fiber alignment in injured tendons. Proteomics studies on the actions of 2DG on tendon healing demonstrated that 2DG stimulated the actin cytoskeletal signaling in injured tendons. Unexpectedly, we found that the mid- substance of tendons expressed muscle type myosin II components but a strongly down-regulated expression of these molecules when injured, whereas 2DG restored these changes. Furthermore, injured tendon-derived progenitor cells responded to 2DG, up-regulated expression of muscle type myosin light chains, and rearranged actin cytoskeletons. Previous studies have demonstrated that actin cytoskeletal organization and myosin II activity are required for collagen fiber alignment. Taken together, we hypothesize that upregulation of muscle type myosin II molecules in injured tendons may stimulate collagen fiber alignment. Furthermore, that glucose controls the expression of muscle type myosin II molecules and the actin cytoskeletal organization. To test these hypotheses, we propose two aims. In Aim 1, we will determine alterations of myosin II molecules in tendons during injury, healing, and regeneration. Spatiotemporal changes in the myosin II components (myosin heavy and light chains) will be defined in injured tendons using four mouse Achilles tendon injury models. In Aim 2, we will determine the roles of muscle type of myosin II in injured tendon-derived cells (inTPCs). InTPCs will be treated with 2DG in mono- or 3D-culture, and changes in the expression of myosin II molecules, RhoA/Rac activity, collagen fiber alignment, and mechanical properties will be examined. In addition, in the same model, the effects of gain- and loss-of-function of muscle myosin II molecules will be determined. Best of our knowledge, it has not been known whether muscle and non-muscle myosin II molecules have an exclusive function in tendons. Thus, this project fits into the scope of R21, and the outcome provides the first clues to answer this question.