Regulation of muscle satellite cell proliferation and differentiation by local trophic factors.

The skeletal muscle is a terminally differentiated tissue. Its capacity to repair following injury or disease depends on a population of myogenic precursors, named satellite cells. These cells are localized beneath the skeletal muscle fiber, in a specialized microenvironment, the niche. The niche preserves the homeostatic conditions of satellite cell quiescence, but at the same time, it ensures their responsiveness to mechanical, physical and chemical triggers from the surrounding environment. Therefore, the composition of the external milieu is critical in determining satellite cell behavior. As a matter of fact, during aging or under pathological conditions, alterations of the extracellular environment entail a severe impairment of satellite cell ability to sustain regeneration and repair of the skeletal muscle tissue. The general goal of this thesis was to focus on some of the trophic factors potentially present in the satellite cell niche in vivo and to characterize their role on the modulation of satellite cell functions in vitro. The first part of the research activity dealt with the study of the trophic effect of ATP on mouse myoblast proliferation. From literature, it emerged that ATP is a potential regulator of the skeletal muscle regenerative program, however the signalling mechanism remained partially unknown. We observed that ATP increased myoblast growth rate, effect that was mimicked by low concentrations of H2O2. Reactive oxygen species (ROS) imaging revealed that ATP induced H2O2 production, at concentrations comparable to those effective in triggering myoblast proliferation. Interestingly, the exposure to equimolar concentrations of adenosine did not mimic the effect of ATP, excluding any role for the main hydrolysis product of ATP in the control of cell cycling. This result was in agreement with data reporting that the specific enzymes responsible for ATP degradation are poorly expressed in myoblasts and become upregulated after cell differentiation. In line with the latter observation, it appeared reasonable that the differentiating skeletal muscle cells were more exposed to ATP-derived adenosine than proliferating myoblasts, and this suggested a potential physiological role for the nucleoside adenosine in the later phases of myogenesis. Taking into account that adenosine receptors (ARs) are present in mouse myotubes, in a second study we hypothesized a crosstalk between nAChRs and ARs. Using the Ca2+-imaging technique, we observed that the pharmacological modulation of ARs triggered variations in the nAChR-driven ([Ca2+]i) spikes. Moreover, our preliminary results suggest not only an interplay between the two receptors but also that endogenous adenosine is tonically released by twitching myotubes and activates its receptors. The third research project was aimed at exploring the role of neural agrin, a heparan sulphate proteoglycan, so far known as the key organizer of post-synaptic elements during skeletal muscle differentiation/regeneration. Besides agrin's canonical effect on the maturation of the NMJ, novel roles have been discovered in the recent years, suggesting that the neurotrophic factor has pleiotropic effects. In this new context, we pursued the identification of potential new roles for neural agrin in the determination of satellite cell behaviour. Firstly, the analysis of different cell models, including C2C12 cell line and primary mouse and human cells, and revealed an increase in IL-6 secretion following exposure to agrin. Secondly, we addressed the hypothesis of agrin as a potential modulator of human myoblasts proliferation. Our preliminary results demonstrate that agrin enhances the proliferative capacity of human satellite cells and suggest the potential mechanism involved in the signaling cascade.