Perturbation of muscle stem cell differentiation by small molecules

Skeletal muscle is one of the most important and plastic tissues of our body. Damaged or stressed skeletal muscle undergoes biological repair and formation of new myofibers upon regeneration signals that activate a complex cross talk between heterogeneous populations of muscle mononucleate cells. The result of this dynamic interplay is the activation of a specialized population of myogenic progenitors, the satellite cells (SCs). Satellite cells are mitotically quiescent and upon activation by regenerative signals they can divide asymmetrically and give rise to myogenic cells (myoblasts) that proliferate, differentiate and fuse to pre-existing myofibers or form new myofibers. The cell context plays an important role in maintaining the satellite cells in the quiescent state and in activating them following regeneration cues. These context dependent effects are often referred to as the stem cell niche. This doctoral thesis aims at understanding the molecular mechanisms that control the activation process and the differentiation decisions involved in the muscle regeneration process. To this end, we performed a highthroughput screening of modulators of differentiation by using the technology of automated fluorescence microscopy. We screened the collection of small molecules of the Prestwick library, containing the drugs approved by the U.S Food and Drug Administration (FDA). We developed fluorescence microscopy readouts in order to monitor the differentiation of mesoangioblasts (MABs), a muscle pluripotent cell line, and of a heterogeneous mix of muscle mononucleate cells. The readouts were designed to monitor differentiation in three different directions, skeletal muscle, adipogenic progenitors and osteoblasts. To date we have screened 560 compounds and we generated a list of “hit” molecules that can perturb the differentiation potential of the cells in the three lineages. We have performed secondary experiments to validate the list of putative interfering small molecules and, in parallel, we have built a similarity tree from a collection of transcriptional expression profile data from cultured human cells treated with bioactive small molecules, developed by the Connectivity Map team in the Broad Institute of MIT. In the second part of this thesis, we decided to further investigate the role of metformin, a drug used in the treatment of diabetes type II, in skeletal muscle differentiation. Even though metformin was one of the screened compounds that did not exhibit perturbation of the differentiation readoutswe were interested in its role in skeletal muscle differentiation since it is implicated in perturbations of the metabolism. Previous studies had suggested that muscle regeneration is improved upon AMPK activation. Short-term calorie restriction enhances the number and the myogenic potential of satellite cells in the muscles of young and old mice with an associated increase in mitochondrial abundance and an enhancement of transplant efficiency. Moreover, different metabolic pathways have been described to be involved in the establishment of the quiescent state. In pathological conditions, as in Duchenne muscular dystrophy (DMD), the repeated cycles of muscle degeneration-regeneration use up the satellite cell pool and decrease their regeneration potential rendering return to quiescence a crucial step in the regeneration process. Recently it has been reported that mTORC1 activity is necessary for the transition of satellite cells from a quiescent G0 phase to a quiescent G-alert phase, characterized by elevated propensity to cycle, increased mitochondrial activity and enhanced myogenic differentiation. Building on the above observations, I asked whether metformin, a calorie restriction-mimicking drug, could affect the activation, proliferation and differentiation of myoblasts in vitro (C2C12 cell line) and of satellite cells in vivo. Our results show that metformin reduces C2C12 myoblasts growth and inhibits their myogenic differentiation in the absence of apoptosis. This inhibition is accompanied by a reversible delay of the cell cycle in the G2/M phase, incompatible with terminal differentiation. When added in terminally differentiated myotubes, metformin induces their degradation and atrophy. Moreover, we demonstrated that metformin delays satellite cell differentiation by postponing exit from quiescence and cell cycle entry. The delayed activation of satellite cells was followed by belated regeneration of skeletal muscle after cardiotoxin injury and was associated with mTOR inhibition and reduced RPS6 phosphorylation. Since the activation, the asymmetric division and differentiation of satellite cells are a prerequisite for the regeneration of the skeletal muscle, metformin treatment could be a way to control the balance between the diverse stem cell fates.