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Ca 2+ signaling plays a key role during human myoblast differentiation. Among Ca 2+ -sensitive pathways, calcineurin is essential for myoblast differentiation and muscle regeneration. Nuclear factor of activated T-cell (NFAT) transcription factors are the major calcineurin targets. We investigated the expression and the role of each NFAT gene during human primary myoblast differentiation. We found that three NFAT isoforms are present, NFATc1, NFATc3 and NFATc4. Importantly, while their mRNA expression increases during differentiation, NFATc1 is more highly expressed in myotubes, whilst NFATc4 is specifically maintained in reserve cells. NFATc3 is present in both cell types, although no specific role during myoblast differentiation was observed. Knockdown of either NFATc1 or NFATc4 affects the differentiation process similarly, by decreasing the expression of late differentiation markers, but impairs myotube formation differently. Whereas NFATc1 knockdown strongly reduced the number and the surface area of myotubes, NFATc4 knockdown increased the surface area of myotubes and reduced the pool of reserve cells. We conclude that NFAT genes have specific roles in myotube formation and in the maintenance of the reserve cell pool during human postnatal myogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

STIM proteins populate and expand cortical endoplasmic reticulum (ER) sheets to mediate store-operated Ca(2+) entry (SOCE) by trapping and gating Orai channels in ER-plasma membrane clusters. A longer splice variant, STIM1L, forms permanent ER-plasma membrane clusters and mediates rapid Ca(2+) influx in muscle. Here, we used electron microscopy, total internal reflection fluorescence (TIRF) microscopy and Ca(2+) imaging to establish the trafficking and signaling properties of the two STIM1 isoforms in Stim1(-/-)/Stim2(-/-) fibroblasts. Unlike STIM1, STIM1L was poorly recruited into ER-plasma membrane clusters and did not mediate store-dependent expansion of cortical ER cisternae. Removal of the STIM1 lysine-rich tail prevented store-dependent cluster enlargement, whereas inhibition of cytosolic Ca(2+) elevations or removal of the STIM1L actin-binding domain had no impact on cluster expansion. Finally, STIM1L restored robust but not accelerated SOCE and clustered with Orai1 channels more slowly than STIM1 following store depletion. These results indicate that STIM1L does not mediate rapid SOCE but can trap and gate Orai1 channels efficiently without remodeling cortical ER cisternae. The ability of STIM proteins to induce cortical ER formation is dispensable for SOCE and requires the lysine-rich tail of STIM1 involved in binding to phosphoinositides., (© 2015. Published by The Company of Biologists Ltd.)

Myogenesis involves expression of muscle-specific transcription factors such as myogenin and myocyte enhancer factor 2 (MEF2), and is essentially regulated by fluctuations of cytosolic Ca(2+) concentration. Recently we demonstrated that molecular players of store-operated Ca(2+) entry (SOCE), stromal interacting molecule (STIM) and Orai, were fundamental in the differentiation process of post-natal human myoblasts. Besides STIM and Orai proteins, the family of transient receptor potential canonical (TRPC) channels was shown to be part of SOCE in several cellular systems. In the present study, we investigated the role of TRPC channels in the human myogenesis process. We demonstrate, using an siRNA strategy or dominant negative TRPC overexpression, that TRPC1 and TRPC4 participate in SOCE, are necessary for MEF2 expression, and allow the fusion process to generate myotubes of normal size. Conversely, the overexpression of STIM1 with TRPC4 or TRPC1 increased SOCE, accelerated myoblast fusion, and produced hypertrophic myotubes. Interestingly, in cells depleted of TRPC1 or TRPC4, the normalization of SOCE by increasing the extracellular calcium concentration or by overexpressing STIM1 or Orai1 was not sufficient to restore normal fusion process. A normal differentiation occurred only when TRPC channel was re-expressed. These findings indicate that Ca(2+) entry mediated specifically by TRPC1 and TRPC4 allow the formation of normal-sized myotubes.