RNA sequencing and immunofluorescence of the myotendinous junction of mature horses and humans.

The research attempted to identify previously unknown components of the myotendinous junction (MTJ) in mature animals and humans. The outcome revealed the expression of several unexplored genes linked with the MTJ, contributing to diverse biological processes.

Methodology

• The study used samples from the superficial digital flexor (SDF), a muscle-tendon interface, obtained from 20 horses. The tissue was meticulously split into muscle, MTJ, and tendon fractions through a sequential cryosectioning approach.
• RT-PCR, a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets, was performed for genes known to express in these tissue fractions.
• t-Distributed Stochastic Neighbor Embedding (t-SNE) plots were used to pick samples from five horses for RNA sequencing. This is a statistical method for imagining high-dimensional data by giving each datapoint a location in a two or three-dimensional map.
• The expressions of genes identified through RNA sequencing were studied through immunofluorescence, a technique used for light microscopy with a fluorescence microscope, on human hamstring MTJ tissue.

Findings

• The primary finding of the study was that RNA sequencing identified the expression of a panel of 61 genes enriched at the MTJ. This included 48 previously unknown genes for the MTJ and 13 genes which had previously been reported to associate with the MTJ.
• Through immunofluorescence on tissue sections of human MTJ, researchers were able to confirm the expression of both known and previously undescribed MTJ genes at the protein level. Known genes included COL22A1 (collagen XXII), NCAM (neural cell adhesion molecule), POSTN (periostin), NES (nestin), OSTN (musclin/osteocrin), whereas novel genes included MNS1 (meiosis-specific nuclear structural protein 1), and LCT (lactase).

Conclusion

• In conclusion, the research identified previously unknown MTJ genes in muscle-tendon interface tissues enriched with myofiber tips. This discovery is significant because these genes are part of diverse biological processes which could be vital for the maintenance of the specialized MTJ, which is central in the transmission of high forces between muscle and tendon.