Comparative Analysis of the Extracellular Matrix Proteome across the Myotendinous Junction

The
myotendinous junction is a highly interdigitated interface
designed to transfer muscle-generated force to tendon. Understanding
how this interface is formed and organized, as well as identifying
tendon- and muscle-specific extracellular matrix (ECM), is critical
for designing effective regenerative therapies to restore functionality
to damaged muscle–tendon units. However, a comparative analysis
of the ECM proteome across this interface has not been conducted.
The goal of this study was to resolve the distribution of ECM proteins
that are uniformly expressed as well as those specific to each of
the muscle, tendon, and junction tissues. The soleus muscles from
5-month-old wild-type C57BL/6 mice were harvested and dissected into
the central muscle (M) away from tendon, the junction between muscle
and tendon (J) and the tendon (T). Tissues were processed by either
homogenizing in guanidine hydrochloride or fractionating to isolate
the ECM from more soluble intracellular components and then analyzed
using liquid chromatographytandem mass spectrometry. Overall,
we found that both tissue processing methods generated similar ECM
profiles. Many ECM were found across the muscle–tendon unit,
including type I collagen and associated fibril-regulating proteins.
The ECM identified exclusively in M were primarily related to the
basal lamina, whereas those specific to T and J tissue included thrombospondins
and other matricellular ECM. Type XXII collagen (COL22A1) was restricted
to J, and we identified COL5A3 as a potential marker of the muscle–tendon
interface. Immunohistochemical analysis of key proteins confirmed
the restriction of some basal lamina proteins to M, tenascin-C to
T, and COL22A1 to J. COL5A3, PRELP, and POSTN were visualized in the
tissue surrounding the junction, suggesting that these proteins play
a role in stabilizing the interface. This comparative map provides
a guide for tissue-specific ECM that can facilitate the spatial visualization
of M, J, and T tissues and inform musculoskeletal regenerative therapies.

肌肌腱连接（myotendinous junction）是一种高度交错的特化界面，其核心功能是将肌肉产生的收缩力传递至肌腱。阐明该界面的形成与组织构建机制，并鉴定肌腱与肌肉特异性细胞外基质（extracellular matrix, ECM），对于开发能够修复受损肌-肌腱单元、恢复其功能的高效再生疗法至关重要。然而，目前尚未有针对该界面处细胞外基质蛋白质组开展比较分析的研究。本研究旨在解析在肌肉、肌腱及连接组织中均一表达的细胞外基质蛋白，以及各组织特异性细胞外基质蛋白的分布特征。研究人员采集了5月龄野生型C57BL/6小鼠的比目鱼肌，并将其解剖分离为远离肌腱的中央肌肉组织（M）、肌-肌腱连接组织（J）与肌腱组织（T）。随后采用两种方式处理组织：一是在盐酸胍中进行匀浆破碎，二是通过分级分离法从可溶性更强的细胞内组分中纯化得到细胞外基质，之后借助液相色谱-串联质谱进行分析。整体而言，两种组织处理方法得到的细胞外基质表达谱高度相似。在整个肌-肌腱单元中均检测到多种细胞外基质蛋白，包括I型胶原及其相关的纤维调节蛋白。仅在M组织中特异性表达的细胞外基质蛋白主要与基底膜相关，而T与J组织特异性的细胞外基质蛋白则包含血小板反应蛋白家族及其他基质细胞蛋白。XXII型胶原（COL22A1）仅局限表达于J组织，研究同时鉴定出COL5A3可作为肌-肌腱连接界面的潜在标志物。针对关键蛋白的免疫组织化学分析证实，部分基底膜蛋白仅表达于M组织，腱生蛋白-C（tenascin-C）仅表达于T组织，而COL22A1仅表达于J组织。在连接组织周围的区域中可观测到COL5A3、PRELP及POSTN的表达，这提示上述蛋白在稳定肌-肌腱连接界面的过程中发挥着重要作用。本研究构建的比较图谱为组织特异性细胞外基质提供了精准参考，可助力M、J及T组织的空间可视化研究，并为肌肉骨骼再生疗法的开发提供关键指导。