Understanding and controlling the mechanisms that regulate muscle stem cell trajectories in the differentiation hyperspace using small molecules

Skeletal muscle is one of the most abundant tissues in the human body and it is responsible for the generation of voluntary movements and the control of body metabolism. Due its role and location, overstretching, trauma and excessive straining are responsible for muscle damage which is counterbalanced by considerable regenerative capabilities to preserve muscle function over time. Skeletal muscle regeneration requires the complex interplay between myogenic progenitors called Satellite cells (MuSC) and the surrounding interstitial cells whose activity is strictly coordinated by the inflamed environment of the damaged muscle. In Duchenne muscular dystrophy (DMD), the mutation in the dystrophin gene affects the tissue structural stability resulting in muscle damage caused by the daily wear and tear. In this context the early stage regenerative response that counterbalances muscle damage is replaced over time by fibrotic and fat tissues infiltration impairing muscle function. Among the interstitial cell populations, fibro Adipogenic Progenitors (FAPs) and vessel-associated myogenic progenitors (mesoangioblasts) are subjects of increasing interest. FAPs are considered as the source of fibrotic and fat tissue infiltrations concomitant with muscular dystrophy while mesoangioblasts are multipotent stem cells with the ability to differentiate in many mesoderm cell type including myocytes. This doctoral thesis aims at identifying and characterizing new compounds able to reshape the differentiation trajectories of these multipotent stem cells in order to counterbalance muscle impairment associated with pathological conditions. The identification of compounds able to increase mesoangioblasts (Mabs) myogenic potential or to decrease FAPs propensity to differentiate into adipose and fibrotic tissues would be of great relevance to counteract the consequences of muscle myopathies. To this end we used FAPs isolated from mdx mice (a model for DMD) and Mabs to perform two High-Content Screenings that can be employed to reshape and control the differentiation trajectories under small-molecule perturbation. We used automated fluorescence microscopy to monitor Mabs differentiation into three different lineages, (i.e., skeletal muscle, adipogenic progenitors and osteoblasts), promoted by small-molecules induction. Moreover, we used mdx FAPs, which spontaneously differentiate into adipocytes and fibroblasts in vitro, to identify small molecules able to decrease their differentiation potential. We have screened 560 compounds on Mabs and more than 5000 compounds on FAPs and we generated a list of “hit” molecules that can perturb the differentiation potential of these cells. Moreover, we have characterized different glucocorticoids for their ability to alter FAPs differentiation potential. Among them, while Budesonide inhibits adipogenesis, Halcinonide and Clobetasol show potential as drugs to inhibit fibrogenesis. Since these compounds are already used as drugs, although for the treatment of other disorders, their ability to impair FAPs differentiation could be potentially useful to counteract muscle degeneration in DMD. In the context of skeletal muscle differentiation and mesoangioblasts fate decision, the mitogen-activated protein kinases P38α/β, play a central role. P38α/β are involved in the regulation of MuSC activation, asymmetric division and their terminal differentiation in mature myotubes. Despite P38α/β importance in the regulation of these processes, the mechanisms behind its activation are still debated. Both cell-to-cell contact and TNF prime P38α/β phosphorylation and activation during myogenesis. Cell adhesion molecule-related/down-regulated by oncogenes (Cdo), a multifunctional surface protein, has been implicated in myogenesis. Following cell-to-cell contact and cadherin ligation, Cdo binds C-Junamino-terminal kinase-interacting protein 4 (JLP) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 (Bnip-2) and both act as scaffolds for recruitment of P38 and Cdc42. The formation of this complex leads to the activation of Cdc42. Cdc42 switch from GDP to GTP results in the activation of the phosphorylation cascade leading to P38α/β activation. However, the molecular players involved in the signal transduction between the activated Cdc42 and the MAPK cascade are still not known. We focused on p21-activated kinase 1 (PAK1) a member of the p21 activated kinase family, which is a downstream effector of Cdc42. We found that the PAK1 inhibitor IPA-3 reduces P38α/β phosphorylation and myogenin expression during Mabs differentiation and this inhibition is accompanied by a delayed exit from the cell cycle. We demonstrated that IPA-3 treatment ultimately inhibits myogenic differentiation of Mabs, C2C12 cells and MuSC. Moreover, we found that mice treated with IPA-3 have a delayed recovery from cardiotoxin-induced muscle injury. These results support the hypothesis that PAK1 is involved in the in vivo myogenic differentiation program. We can speculate that P38α/β phosphorylation impairment by PAK1 inhibition, leading to regenerative impairment due to cell cycle exit failure, could drive an accumulation of activated satellite cells spared by the first round of regeneration but ready for the next one. Thus representing a considerable outcome as therapeutic application in muscle dystrophy in order to hinder and retard satellite cells exhaustion in muscular dystrophy affected patient.