Titolo della tesi: X-MET: a bioengineered muscle construct as a model to study muscle physiopathology
Skeletal muscle disorders are characterized by the loss of muscle mass and function, which cannot be effectively evaluated using conventional two-dimensional (2D) in vitro cultures. In vivo models, while informative, require large numbers of animals and involve time-consuming experiments to study molecular processes at various disease stages. To overcome these limitations, research has focused on developing bioengineered three-dimensional (3D) models that closely mimic the complex architecture of muscle and allow for accurate assessment of key functional characteristics.Complying with these principles, in our laboratories it has been developed a scaffold-free 3D engineered skeletal muscle tissue from murine primary cultures, namely, ex vivo muscle engineered tissue (X-MET) (Carosio et al. 2013). The X-MET recapitulates, in vitro, morphological, molecular and functional characteristics of skeletal muscle: it is able to contract spontaneously as well as to respond to electrical stimulation. The general aim of this thesis’s project is to define the versatility of X-MET in replicating the intricate structural and functional complexities of skeletal muscle in vitro. Specifically, we sought to simulate two form of skeletal muscle loss, cancer associated cachexia and muscular dystrophy, to validate X-MET’s capability to accurately mimic both acquired and congenital diseases leading to muscle wasting and ultimately to test the efficacy of therapeutic compounds. At first, we employed X-MET to model cancer-associated cachexia and to study the effectiveness of selective inhibition of IL-6 trans-signalling in counteracting the cachectic phenotype. Conditioned medium (CM) derived from C26 adenocarcinoma cells was used as a source of soluble factors contributing to the establishment of cancer cachexia. A dose of 1.2 ng/mL of glycoprotein-130 fused chimaera (gp130Fc) was added to cachectic culture medium to neutralize IL-6 trans-signalling. Concurrently, microtissue derived from MDX4cv mice was employed to replicate dystrophic conditions, the spontaneous contractility of the X-MET ensured the induction of the characteristic contraction-associated damage critical in dystrophy progression. Using this system, we also investigated the role of muscle-specific pathological secretions, such EVs secreted by dystrophic muscle, in disease progression. Molecular, histological and functional analyses were conducted to evaluate disease-specific markers and pathological mechanisms. Our data demonstrate that X-MET model successfully replicated key hallmarks of cancer-associated cachexia, including muscle atrophy and altered metabolic profiles, upon exposure to C26 adenocarcinoma cell-derived medium demonstrating the ability of the in vitro tissue to mimic muscle response to environmental cues. Similarly, constructs obtained from MDX4cv mice exhibited characteristics of muscular dystrophy, such as sarcolemma damage and impaired regeneration, validating our model’s ability to simulate intrinsic muscle dysfunctions. Overall, both in vitro designs covered herein establish the X-MET model as a robust and versatile platform for studying skeletal muscle disorders in vitro, allowing a rapid and accurate analysis, reducing experimental time/costs, and limiting the number of animals. This 3D in vitro system not only aid in unravelling the molecular basis of skeletal muscle diseases but also provide a valuable platform for in vitro drug testing.