Towards a deep knowledge of the extracellular matrix proteins functions in the Duchenne Muscular Dystrophy

Project Information

MYO_MATRIX

Grant agreement ID: 101105479

DOI

10.3030/101105479

Project closed

EC signature date 12 April 2023

Start date 1 January 2024

End date 31 March 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 206 859,39

Coordinated by

UNIVERSITE LYON 1 CLAUDE BERNARD
France

Periodic Reporting for period 1 - MYO_MATRIX (Towards a deep knowledge of the extracellular matrix proteins functions in the Duchenne Muscular Dystrophy)

Reporting period: 2024-01-01 to 2026-03-31

Summary of the context and overall objectives of the project

Adult healthy skeletal muscle has a powerful capacity to completely regenerate after an injury. On the opposite, muscular dystrophies (MDs) are devastating diseases where the regenerative capacities are overwhelmed and the muscle fibers are progressively replaced by fibrosis, leading to muscle loss of function and, finally to death, due to respiratory and cardiac failures. Since muscle stem cells are key factors in skeletal muscle regeneration, studies have mainly focused on the properties of these cells, as well as on their regulation by the neighboring cells. Despite the fact that excessive deposition of extracellular matrix (ECM) is the main cause of the loss of muscle function in MDs, investigation of ECM roles in muscle homeostasis remains scarce. The reason is that fibrosis is envisaged as one of the final outcomes of the disease. However, recent investigations in other tissues showed that ECM is an active partner of cells, and which dynamics is necessary for tissue remodeling. The project aimed to define the molecular ECM determinants at work during degenerative myopathies. The approach was to compare regenerative ECM (from healthy muscle undergoing regeneration) and dystrophic ECM (from diseased muscle) through proteomic-based mass spectrometry methodologies after obtaining sk-ECM from various mouse models by exploiting decellularization methods. The comparison between healthy regenerating (remodeling sk-ECM) and dystrophic (fibrotic sk-ECM) muscles allowed the identification of new molecular regulations from the ECM to various cell types in the muscle, notably the fibroadipogenic precursor cells, which are responsible for fibrosis.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project investigated the molecular mechanisms underlying extracellular matrix (ECM) and fibroblastic differentiation in Duchenne muscular dystrophy (DMD), with particular focus on fibro-adipogenic progenitors (FAPs) and molecules identified via proteomic analysis of the ECM. A major component of the work involved the optimization and comparison of skeletal muscle decellularization strategies to improve ECM preservation and enrichment for downstream proteomic analyses. Histological and biochemical evaluations enabled the identification of the most efficient approach for reducing cellular contamination while maintaining ECM integrity, thereby improving the extraction and characterization of matrisome and matrix-associated proteins from dystrophic skeletal muscle. Subsequently, advanced mass spectrometry-based proteomic analyses were performed to characterize the dystrophic skeletal muscle matrisome and identify differentially regulated proteins associated with disease progression. These analyses revealed extensive ECM remodeling and identified AnxA2 as a significantly dysregulated protein in dystrophic muscle. The project further investigated the biological relevance of several molecules through biochemical, molecular docking, bioinformatic, cell culture and imaging approaches. Experimental analyses confirmed the functional involvement of two ECM molecules in the fibrotic and adipogenic behavior of FAPs, respectively. The downregulation of expression, or the change in their post-translational modification (phosphorylation) of these two molecules directly stimulates the differentiation of FAPs in to fibrogenic or andipogenic cells. In vitro functional experiments allowed to rescue the phenotype on FAP by adding the molecules to the dystrophic ECM.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

A key advancement beyond the current state of the art was the integration of optimized ECM decellularization approaches with proteomic profiling to achieve improved characterization of the dystrophic skeletal muscle matrisome. This strategy enabled the identification of molecular alterations that may not be detectable using conventional tissue proteomics approaches. Importantly, the study identified two ECM components as potential regulators of fibroblastic differentiation within the dystrophic microenvironment. Interestingly, the regulation of these molecules in the dystrophic ECM relates on their post-translational modification or the decrease of their presence in the ECM. These findings introduce new mechanistic perspectives linking ECM components and the regulation of tissue homeostasis in a chronic disease.