"Time window" effect of Yoda1-evoked Piezo1 channel activity during mouse skeletal muscle differentiation

Abstract

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) and on adult muscle fibres.

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 myofibres and at the adult neuromuscular junction.

Results: Piezo1 channels were detected by immunostaining in satellite cells (SCs) and myotubes as well as in adult myofibres. In the skeletal muscle precursors, Yoda1 treatment stimulated the differentiation and cell fusion rather than the proliferation of SCs. Moreover, in myotubes, Yoda1 induced significant [Ca²⁺ ]_i transients, without detectable [Ca²⁺ ]_i response in adult myofibres. 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 myofibres.

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.

Keywords: Piezo1 channels; Yoda1; myogenesis; myotubes; satellite cells; skeletal muscle myofibres.

FIGURE 1

Localization of Piezo1 channels in skeletal muscle cells by confocal microscopy. A, Immunostaining for Piezo1 channels in a Pax7‐positive satellite cell (SC) (arrow) attached onto a FDB fibre (24 h post plating, 50 SCs were analysed). Note the close by Pax7‐negative nucleus likely belonging to the skeletal muscle fibre. B, Representative Piezo1 channel clusters detected in differentiating myotubes (7 day post plating). C, Immunostaining revealed Piezo1 channels organized in scattered clusters also in myofibres. Note the different scale bars in (B) and (C). D, Comparison of cluster length detected in myotubes and myofibres (n = 32 and 451 clusters analysed in myotubes and myofibres; ^‡ P < .0001, Mann–Whitney test). E, Localization of Piezo1 channel clusters at a nearby endplate region of a myofibre (24 h post plating). Scale bars: 10 μm in (A) and 20 μm in (B), (C) and (E). Each group of confocal experiments were carried out on at least three independent cell culture preparations

FIGURE 2

Effect of Yoda1 on Pax7‐positive cell numbers. A, Pax7‐positive satellite cells (SCs) after 48 h from the dissociation, in control conditions and in the presence of Yoda1 (3 and 10 μM). Scale bars: 50 μm. B, The cell treatment with 3 or 10 μM did not affect the percentage of Pax7‐positive cells (control: n = 131; 3 μM: n = 137; 10 μM: n = 29 optical fields; P > .05, ANOVA, Dunnett's post hoc test). Experimental replicates n = 4; data from five independent experiments

FIGURE 3

Dose–response effect of Yoda1 on myogenic precursors. A, Representative images and graph summarizing the percentage of MyoG‐positive cells cultured in differentiation medium for 72 h in controls and in the presence of different Yoda1 concentrations (control: n = 64 optical fields; 0.5 μM: n = 38; 3 μM: n = 62; 5 μM: n = 46; 10 μM: n = 37, ^† P < .01, ANOVA, Dunnett's post hoc test). B, Effect of Yoda1 on cell morphology (control: n = 192 optical fields; 0.5 μM: n = 185; 3 μM: n = 293; 5 μM: n = 244; 10 μM: n = 175, ^* P < .05, ^† P < .01 and ^‡ P < .001, ANOVA, Dunnett's post hoc test) after a treatment of 72 h. Experimental replicates n = 2; data from two independent experiments

FIGURE 4

Yoda1 affected myocyte orientation. A, Representative 8‐bit greyscale images of MyoG‐positive cells cultured in differentiation medium for 72 h and, below, the corresponding analysis of the mean cell orientation using the FiberFit software. Scale bar: 50 μm. B, On the left, the dispersion parameter k, which quantifies the degree of cell alignment (for further details, see Section 4) in control condition (n = 69 cells) and in cells treated with Yoda1 (3 μM) (n = 61 cells; ^† P = .0089, t test). On the right, the goodness of fit R ² (P = .14, Mann–Whitney test). Control and treated cells were from different culture dishes. Experimental replicates n = 3; data from two independent experiments. C, The presence of Yoda1 (3 μM) increased the number of multinucleated desmin‐positive cells (control: 24.38% ± 1.85%, n = 42 optical fields; Yoda1: 33.53% ± 2.28% , n = 53 optical fields; ^‡ P = .0034, t test). Experimental replicates n = 3; data from four independent experiments

FIGURE 5

Effect of Yoda1 on adult skeletal muscles. A, Representative recording traces of multi quantal endplate currents (EPCs) in the absence (Control) and presence of Yoda1 (5 μM). B, The real dynamics of multi quantal EPC amplitude changes during recording in a separate neuromuscular junction. C, Representative recording traces of miniature endplate currents (MEPCs) in the absence (Control) and presence of Yoda1 (5 μM). D, Averages of the EPC and MEPC amplitudes and MEPC frequencies in absence and in presence of Yoda1 (5 and 10 μM) expressed as percentage respect to controls (Ctrl); n = 6 animals, paired t test. E, Example of tetanic contraction before and after Yoda1 application (5 μM). F, Averages of tetanus force recorded before and after Yoda1 (5 and 10 μM) expressed as percentage respect to controls (Ctrl); n = 6 animals, paired t test

FIGURE 6

Effect of Yoda‐1 on [Ca²⁺] _i . A, In flexor digitorum brevis (FDB) myofibres, Yoda1 (10 μM) did not alter the basal [Ca²⁺] _i . The [Ca²⁺] _i variations induced by high K⁺ depolarisation (KCl 60 mM) and caffeine (Caff 40 mM) were used as positive controls of cell responsiveness to stimuli able to increase the [Ca²⁺] _i . B, In myotubes, at the same concentration, the agonist of Piezo1 channels induced [Ca²⁺] _i oscillations in 35% of the observed myotubes (see text for further details). The effect of Yoda1 was prevented by myotube preincubation with Gd³⁺ (300 μM). Each set of Ca²⁺ imaging experiments was carried out on three independent cell culture preparations