Dissecting the role of Collagen VI at the myotendinous junction by exploiting a mouse model of ColVI-related disorders

Collagen VI (ColVI) is an extracellular matrix (ECM) protein mainly expressed by fibroblasts and known to be expressed in a plethora of tissues. Although it belongs to the collagen family of proteins, its structure is unique in terms of supramolecular assembly, as ColVI tetramers assemble in a beaded microfilament network once secreted into the ECM. Due to its complex assembly (a ColVI monomer is a heterotrimer made of three different α-chains) and its ability to bind several ECM proteins but also membrane-bound receptors, ColVI has many biological functions. ColVI chains are mostly known for being affected in human diseases of the musculoskeletal apparatus. Mutations affecting the assembly, secretion or formation of the beaded microfilament network in the ECM of ColVI results in a mild to severe muscular dystrophy, affecting muscles, tendons, cartilage and bones. Based on the type and severity of symptoms, the diseases caused by mutations in COL6A1, COL6A2, COL6A3 and COL6A6 genes are classified as Bethlem Myopathy (BM), Ullrich Congenital Muscular Distrophy (UCMD) or Myosclerosis Myopathy (MM), different types of ColVI related disorders (ColVI-RD). The underlying pathological mechanisms of disease have been investigated thanks to the use of in vitro models derived from patients’ biopsies or in vivo by generating transgenic mouse lines. In particular, the Col6a1-/- mice (ColVI KO) proved to be a useful loss-of-function model of Bethlem Myopathy, due to its complete incapability of producing ColVI. Thanks to this model many discoveries have been made, such as the mitochondrial dysfunction and the suppression of autophagy in muscle, both targetable processes to develop interventions in humans. Nonetheless a lot of details regarding non.muscle specific features are still unclear. In this context, my PhD project aimed at characterizing potential pathological changes at the myotendinous junction (MTJ) and their contribution to ColVI-RD pathogenesis in the ColVI KO mouse model. The MTJ is the connection site of muscle myofibers with the tendon ECM, and it is characterized by peculiar morphological features of the myofiber tip (forming digit-like protrusions) but also by its specific ECM composition and cellular niche. Due to the great forces applied at this site, the MTJ can undergo rupture in healthy individuals and its contribution to muscular dystrophy is mostly unknown. ColVI was previously shown to be expressed in the human MTJ, but its role in this site has not been addressed yet in the ColVI KO mouse model. I was able to isolate an MTJ-enriched diaphragm region for proteomic analysis which proved to be crucial to dissect the pathological changes at the MTJ in ColVI KO mice. I was able to detect changes in the ECM composition, but also in the mechanical properties of the MTJ ECM. Additionally, proteomic and imaging approaches highlighted cytoskeletal alterations at the myofiber tip, possibly due to the altered mechanical properties and ECM composition. Indeed, decreased focal adhesion signaling activation was highlighted, together with a decreased interdigitation of the myofiber protrusions as shown by electron microscopy. These changes also involved the activation of the mechanosensitive YAP/TAZ signaling pathway, potentially triggering cell proliferation in response to increased stiffness. In support of a proliferative response to an injury-like insult, target genes of the transcriptional repressor HIC1, involved in maintenance of quiescent mesenchymal progenitor cells, were upregulated in ColVI KO MTJ extracts. Overall, we show for the first time the involvement of MTJ-specific pathological alterations in a mouse model of ColVI-RD. Our results pave the way to further deepen our understanding of pathological changes at the MTJ in muscular dystrophies.