Dottorato in MORFOGENESI E INGEGNERIA TISSUTALE

Titolo della tesi: Characterization of molecular players promoting functional remodelling of the eX vivo muscle engineered tissue (X-MET)

Mechanical stimuli play a fundamental role in the development and maintenance of both skeletal and cardiac muscle. Recent advances in tissue engineering suggest that mechanical tension enhances the structural and functional remodelling of engineered constructs (Riehl et al., 2012). In this context, we demonstrated that passive tension applied to a scaffold-free engineered skeletal muscle construct, named eX-vivo Muscle Engineered Tissue (X-MET), induces functional remodeling toward a cardiac-like phenotype, offering promising foundational insights for novel regeneration strategies (Cosentino et al., 2023). However, the molecular mechanisms underlying this mechanosensitive process remain unclear. In this study, we investigated how passive stretching influences X-MET behaviour, with a focus on the molecular and functional mechanisms underlying this response. Specifically, we aimed to identify the key cellular players and evaluate the contribution of macrophages, immune cells involved in cardiac conduction (Hulsmans et al., 2017), to the stretch-induced X-MET functional remodeling. To this end, X-MET constructs were generated from primary murine muscle culture and subjected (or not) to mechanical tension. Macrophage involvement was assessed via selective depletion. Functional and molecular analyses, including immunostaining, calcium imaging, and biomechanical recordings, were conducted. Our data demonstrated that macrophage population proved essential for the X-MET remodelling process. In their presence, X-MET exhibited increased formation of gap junctions, enhanced Ca²⁺ influx, and improved electrical signal propagation. These effects were markedly reduced when macrophages were depleted, suggesting that they act as mechano-responsive mediators. Further analyses indicate that macrophages may contribute to the mechanosensing ability of the construct by modulating local stiffness and cytoskeletal signaling in response to mechanical cues. Altogether, these findings highlight the role of macrophages as key modulators of mechanically induced tissue adaptation. Beyond its value as a model for studying mechanotransduction, the X-MET construct holds promise as a preclinical platform for gene therapy research. As an additional aim of this study, in collaboration with Prof. Caracciolo's group, we generated X-MET from MDX4cv mice, a widely used Duchenne Muscular Dystrophy (DMD) model, to evaluate the efficacy of non-viral vectors, such as lipid nanoparticles (LNPs), for micro-dystrophin gene delivery. Indeed, while gene therapy for DMD is advancing rapidly (Elangkovan et al., 2021), limitations of adeno-associated virus (AAV) vectors, such as low transgene persistence and immunogenicity, underscore the need for alternatives (Manini et al., 2022). Our results suggest that nanoparticle-based vectors hold great promise, due to their favorable physico-chemical and biological characteristics. Moreover, the X-MET platform provides a physiologically relevant and highly reproducible model to assess delivery efficiency and molecular responses before proceeding to in vivo validation. In summary, this study enhances our understanding of the cellular mechanisms underlying mechanically induced reorganization in engineered muscle tissue and underscores the value of the X-MET platform for investigating muscle remodeling and validating gene delivery strategies.