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.