ISS: Quantifying the Effect of Unloading on Extracellular Matrix Remodeling in the Musculoskeletal System

该项目将研究导致微重力下组织退化的机制。 暴露于太空飞行或地球上减少的机械负荷会导致明显的骨丢失、肌肉萎缩和软组织退化。尽管运动和药理学对策的进展，旨在减少肌肉骨骼恶化在地球上和航天，保护是不完整的，对这些干预措施的反应是可变的。许多研究集中在萎缩和随后恢复的分子和细胞机制上。 然而，很少有人注意到细胞外基质（ECM），细胞驻留的材料。ECM是一种复杂的网络，其组成在特定组织之间变化，取决于机械和功能需求。细胞外基质的变化可以永久改变组织材料的性质，阻止驻留细胞重新建立体内平衡，并影响功能。有必要更好地了解由于微重力而损害组织功能的ECM成分发生的根本变化，在本研究中，将研究ECM蛋白质响应微重力和从微重力恢复后的时间变化，以检验以下假设：肌肉骨骼系统的废用通过减少蛋白质周转和增加酶和非酶交联而不可逆地影响ECM，导致在返回到1G之后功能的不完全恢复。与在几分钟内显著变化的基因表达不同，这是评估微重力生理影响的常用措施，蛋白质组在几小时到几天内发生变化，并提供了一个更稳定的生理指标，可以承受重返，着陆和从国际空间站运送小鼠。将结合代谢标记和蛋白质组学方法，通过向小鼠提供使用内源性细胞机制掺入蛋白质中的非经典氨基酸（ncAA）来跟踪体内ECM周转。ncAA具有生物正交柄，其能够通过与互补化学基团的点击反应来富集新合成的蛋白质。小鼠蛋白质组将在微重力开始后的不同时间点用ncAA标记，以提供响应卸载在体内所有组织中形成的蛋白质的稳健“时间戳”。该研究将首先确定在地球和国际空间站上标记新合成ECM蛋白的最佳参数，然后测试肌肉骨骼ECM对两种不同卸载模式的反应：地球上的后肢悬挂和国际空间站上的太空飞行。伴随研究的三个肌肉骨骼组织：骨，肌腱和骨骼肌，并使用质谱法，将揭示生物标志物和途径，是重要的废用性萎缩和随后的恢复跨肌肉骨骼组织。此外，该研究将为CASIS提供前所未有的资源，通过建立组织共享机制传播ncAA标记的组织，这将使广泛的二级科学研究者受益。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

This project will study the mechanisms that lead to tissue degradation in microgravity. Exposure to spaceflight or reduced mechanical loading on Earth induces marked bone loss, muscle atrophy, and degradation of soft tissues. Despite advances in exercise and pharmacologic countermeasures designed to reduce musculoskeletal deterioration on Earth and in spaceflight, the protection is incomplete and response to these interventions are variable. Many studies have focused on the molecular and cellular mechanisms that underlie atrophy and subsequent recovery. However, little attention has been given to the extracellular matrix (ECM), the material in which cells reside. The ECM is a complex network that varies in composition between specific tissues depending on the mechanical and functional demands. Changes to the ECM can permanently alter tissue material properties, prevent resident cells from re-establishing homeostasis, and affect functionality. There is a need to better understand the fundamental changes that occur in ECM composition due to microgravity that compromise tissue functionality.In this study, temporal changes in ECM proteins in response to, and after recovery from, microgravity will be investigated to test the following hypothesis: disuse of the musculoskeletal system irreversibly affects the ECM by reducing protein turnover and increasing enzymatic and non-enzymatic cross-links, leading to the incomplete restoration of functionality after a return to 1g. Unlike gene expression that significantly varies within minutes, a common measure to assess for the physiological effects of microgravity, the proteome changes over hours to days, and provides a more stable indicator of physiology that can withstand reentry, landing, and transport of mice from the ISS. Metabolic labeling and proteomic methods will be combined to track ECM turnover in vivo by providing mice with non-canonical amino acids (ncAAs) that are incorporated into proteins using endogenous cellular machinery. ncAAs possess bioorthogonal handles that enable the enrichment of newly synthesized proteins through click reactions with complementary chemical groups. The murine proteome will be labeled with ncAAs at different timepoints after the onset of microgravity to provide a robust “time-stamp” of proteins formed in all tissues in the body in response to unloading. The study will first identify the optimal parameters to label newly synthesized ECM proteins on Earth and the ISS, then test response of the musculoskeletal ECM to two different unloading paradigms: hindlimb suspension on Earth and spaceflight on the International Space Station. Concomitant study of three musculoskeletal tissues: bone, tendon, and skeletal muscle, and the use of mass spectrometry, will reveal biomarkers and pathways that are important to disuse atrophy and subsequently recovery across musculoskeletal tissues. In addition, the study will provide an unprecedented resource for CASIS to disseminate ncAA-labeled tissues through established tissue-sharing mechanisms that will benefit a wide range of secondary science investigators.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.