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Aim: Mechanosensitive Piezo1 ion channels emerged recently as important contributors to various vital functions including modulation of the blood supply to skeletal muscles. The specific Piezo1 channel agonist Yoda1 was shown to regulate the tone of blood vessels similarly to physical exercise. However, the direct role of Piezo1 channels in muscle function has been little studied so far. We therefore investigated the action of Yoda1 on the functional state of skeletal muscle precursors (satellite cells and myotubes) as well as on adult muscle fibers. Methods: Immunostaining, electrophysiological intracellular recordings and Ca2+ imaging experiments were performed to localize and assess the effect of the chemical activation of Piezo1 channels with Yoda1, on myogenic precursors, adult myofibers and at the adult neuromuscular junction. Results: Piezo1 channels were detected by immunostaining in satellite cells and myotubes as well as in adult myofibers. In the skeletal muscle precursors, Yoda1 treatment stimulated the differentiation and cell fusion rather than the proliferation of satellite cells. Moreover, in myotubes, Yoda1 induced significant [Ca2+ ]i transients, without detectable [Ca2+ ]i response in adult myofibers. Furthermore, although expression of Piezo1 channels was detected around the muscle endplate region, Yoda1 application did not alter either the nerve-evoked or spontaneous synaptic activity or muscle contractions in adult myofibers. Conclusion: Our data indicate that the chemical activation of Piezo1 channels specifically enhances the differentiation of skeletal muscle precursors, suggesting a possible new strategy to promote muscle regeneration.

Cell membranes, carrying neurotransmitter receptors and ion channels, can be 'microtransplanted' into frog oocytes. This technique allows a direct functional characterization of the original membrane proteins, together with any associated molecules they may have, still embedded in their natural lipid environment. This approach has been previously demonstrated to be very useful to study neurotransmitter receptors and ion channels contained in cell membranes isolated from human brains. Here, we examined the possibility of using the microtransplantation method to study acetylcholine receptors from normal and denervated rat skeletal muscles. We found that the muscle membranes, carrying their fetal or adult acetylcholine receptor isoforms, could be efficiently microtransplanted to the oocyte membrane, making the oocytes become sensitive to acetylcholine. These results show that oocytes injected with skeletal muscle membranes efficiently incorporate functional acetylcholine receptors, thus making the microtransplantation approach a valuable tool to further investigate receptors and ion channels of human muscle diseases.

It is widely accepted that nicotinic acetylcholine receptor (nAChR) channel activity controls myoblast fusion into myotubes during myogenesis. In this study we explored the possible role of nAChR channels after cell fusion in a murine cell model. Using videoimaging techniques we showed that embryonic muscle nAChR channel openings contribute to the spontaneous transients of intracellular concentration of Ca2+ ([Ca2+]i) and to twitches characteristic of developing myotubes before innervation. Moreover, we observed a choline acetyltransferase immunoreactivity in the myotubes and we detected an acetylcholine-like compound in the extracellular solution. Therefore, we suggest that the autocrine activation of nAChR channels gives rise to [Ca2+]i spikes and contractions. Spontaneous openings of the nAChR channels may be an alternative, although less efficient, mechanism. We report also that blocking the nAChRs causes a significant reduction in cell survival, detectable as a decreased number of myotubes in culture. This led us to hypothesize a possible functional role for the autocrine activation of the nAChRs. By triggering mechanical activity, such activation could represent a strategy to ensure the trophism of myotubes in the absence of nerves.

Brain-derived calcium-binding protein S100 induces apoptosis in a significant fraction of rat phaeochromocytoma (PC12) cells. We used single cell techniques (patch clamp, videomicroscopy and immunocytochemistry) to clarify some of the specific aspects of S100-induced apoptosis, the modality(ies) of early intracellular Ca2+ concentration increase and the expression of some classes of genes (c-fos, c-jun, bax, bcl-x, p-15, p-21) known to be implicated in apoptosis of different cells. The results show that S100: (1) causes an increase of [Ca2+]i due to an increased conductance of L-type Ca2+ channels; (2) induces a sustained increase of the Fos levels which is evident since the first time point tested (3 h) and remains elevated until to the last time point (72 h). All these data suggest that S100-derived apoptosis in PC12 cells may be the consequence of a system involving an increase in L-type Ca2+ channel conductance with consequent [Ca2+]i increase which up-regulates, directly or indirectly, the expression of Fos.