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

这个项目将研究在微重力下导致组织降解的机制。暴露在太空中或减少地球上的机械负荷会导致显著的骨丢失、肌肉萎缩和软组织退化。尽管在运动和药物对策方面取得了进展，旨在减少地球和航天中肌肉骨骼的恶化，但这种保护是不完整的，对这些干预措施的反应也是多种多样的。许多研究都集中在萎缩和随后恢复的分子和细胞机制上。然而，细胞外基质(ECM)--细胞所在的物质--却很少受到关注。ECM是一个复杂的网络，根据机械和功能需求，特定组织之间的组成各不相同。细胞外基质的改变可能会永久性地改变组织材料的属性，阻止驻留细胞重新建立内稳态，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组成的根本变化。在本研究中，将研究ECM蛋白质在微重力响应和恢复后的时间变化，以检验以下假设：停用肌肉骨骼系统通过减少蛋白质周转和增加酶和非酶交联来不可逆转地影响ECM，导致恢复到1g后功能的不完全恢复。与基因表达在几分钟内显著变化不同，基因表达是评估微重力生理影响的常见指标，蛋白质组在几个小时到几天内变化，提供了一个更稳定的生理指标，可以承受小鼠从国际空间站的再入、着陆和运输。代谢标记和蛋白质组学方法将结合起来，通过为小鼠提供非规范氨基酸(NCAA)来跟踪体内ECM的周转，NCAA使用内源性细胞机械将其整合到蛋白质中。NCAA拥有生物正交柄，能够通过与互补化学基团的点击反应来丰富新合成的蛋白质。在微重力开始后的不同时间点，将用NCAA标记小鼠蛋白质组，以提供体内所有组织中形成的蛋白质对卸载做出反应的强有力的“时间戳”。这项研究将首先确定标记地球和国际空间站新合成的ECM蛋白质的最佳参数，然后测试肌肉骨骼ECM对两种不同卸载范例的反应：地球上的后肢悬挂和国际空间站上的航天。同时对三种肌肉骨骼组织：骨、肌腱和骨骼肌进行研究，并使用质谱仪，将揭示生物标志物和途径，这些生物标志物和途径对于消除肌肉骨骼组织的萎缩和随后的恢复至关重要。此外，这项研究将为CASIS提供前所未有的资源，通过建立的组织共享机制传播NCAA标记的组织，这将使广泛的二级科学研究人员受益。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.