Characterization of molecular interactions between mononuclear muscle cell populations: the role of Notch signaling

Skeletal muscle regeneration is mediated by a complex crosstalk between several resident mononuclear cell populations. Satellite cells are the main source of new myoblasts and play a pivotal role during the regenerative process. However, their function relies on environmental cues shaped by numerous mononuclear cell populations resident in the skeletal muscle. Fibro-adipogenic progenitors (FAPs) have a prominent role in the regeneration process since they promote myotube formation by positively regulating satellite cell differentiation. However, in pathological conditions, they are responsible for fibrosis and fat infiltrations. Despite the established importance of FAPs in both regeneration and degeneration, the signals that regulate these opposing roles are not fully characterized. Thus, considering the importance of the stem cell niche for maintaining muscle homeostasis, a thorough characterization of the molecular interactions that drive the complex network of cellular crosstalk, and understanding how it is altered in pathological conditions, is of great interest in order to better delineate the muscle regeneration process, and possibly to identify novel therapeutic targets for reversing the negative consequences of muscular disorders. In my PhD thesis I aimed to extend our understanding of the molecular interactions between satellite cells and FAPs using an in vitro co-culture system and focusing on both phenotypic and molecular readouts. By exploiting the Luminex xMAP technology, I aimed to characterize molecules that drive the crosstalk between two populations. I established an antibody panel of 13 molecules that were reported in literature to be involved in muscle regeneration, and tested these molecules in two conditions, growth medium and adipogenic medium. Among the identified molecules, the chemokines SDF-1 and MCP-1 were observed in the secretome of FAPs, indicating a possible novel role of FAPs in homing of satellite cells or macrophages to the site of injury during regeneration. The cross talk between two cell populations is not only achieved by the exchange of soluble molecules, but also by direct cell contact. In particular, I observed a strong inhibition of FAPs adipogenic differentiation when cocultured with satellite cells, thus when the two cells are in physical contact. By contrast, in a transwell co-culture, where cells are physically separated by a porous membrane, I hardly observed any inhibition of adipocyte formation. Since the Notch signaling pathway is responsible for maintaining satellite cells in the quiescent state, thus preventing their differentiation, and is triggered by cell-to-cell contact, we investigated whether the signals modulated by the Notch ligand also affect FAP adipogenesis. To answer this question, FAPs were exposed to the γ-secretase inhibitor, DAPT, or to the Notch ligand, Dll1, an inhibitor and an activator of the Notch pathway respectively. Our results support a conclusion whereby Notch plays an important role in the regulation of FAP adipogenesis in vitro. Moreover, as mentioned before, the direct contact of satellite cell-derived myotubes inhibits FAPs adipogenic differentiation in vitro. Our findings imply that Notch signaling mediates this communication between these two populations. Finally, the involvement of Notch signaling in the regulation of physiological muscle regeneration was addressed in vivo. Inhibition of this pathway during the regeneration process led to mild intramuscular adipocyte accumulation. Overall, our results emphasize the importance of Notch during muscle regeneration not only in modulating myogenesis, but also in restraining the adipogenic potential of fibro-adipogenic progenitors. Interestingly, this mechanism is impaired in FAPs isolated from young dystrophin-deficient, mdx, mice. To test whether this defective regulation of mdx FAPs is due to the cues provided by the inflamed environment triggered by regeneration-degeneration cycles, I treated wild type (wt) mice with cardiotoxin (ctx) in order to induce the regeneration process in an otherwise healthy muscle. I observed a strong inhibition of adipogenic differentiation of FAPs isolated three days after injury. Thus, I conclude that the control mechanism driven by Notch signaling is impaired in a mdx environment and that this impairment is a likely cause of adipose and fibrous infiltrations. To further investigate this phenotypic differences, we performed deep profiling and multiparametric analyses, by exploiting single cell mass cytometry and mass spectrometry based proteomic. Both analyses showed clear differences in the phenotypes of cell populations and proteome profile when wild type and mdx FAPs were compared. In addition the quantification of more than 5000 proteins in FAPs purified from different experimental animals highlights a number of process that are perturbed in the mdx mouse model and could be responsible for the observed insensitivity to stimulation by the Notch ligand.