Résumé :
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Communication n° 137. Myoblast differentiation is a strictly Ca2+-dependent process. We have shown that a main route for Ca2+ entry is through T-type Ca2+ channels. However, because of the importance of cytoplasmic Ca2+ for differentiation, myoblasts have evolved alternative strategies to increase Ca2+. Indeed, when Ni2+ is used to block T-type Ca2+ channels, the differentiation process is slowed down but not fully abolished. The goal of the present work was to assess cytoplasmic Ca2+ requirements during the initial phase of myoblast differentiation by following an early Ca2+-dependent differentiation marker, MEF2. MEF2 expression was evaluated in conditions that prevented either Ca2+ influx from extracellular medium or Ca2+ release from intracellular stores. Fura2 was used to document resting Ca2+ levels, state of filling of Ca2+ stores, and Ca2+ influxes. When external Ca2+ was reduced to 15 µM to abolish influxes, fusion was impeded, but the increase in MEF2 expression was still present which suggests that myoblasts are able to use intracellular Ca2+ stores to differentiate. To confirm this observation, we applied Xestospongin C, an inhibitor of IP3 receptors, in low external Ca2+. In these conditions, MEF2 expression and differentiation was inhibited. Also, when Ca2+ stores were depleted by a low concentration of thapsigargin, MEF2 expression was inhibited. We observed, however, that differentiation still occurred in some myoblasts when low thapsigargin was present. In these myoblasts, differentiation was triggered by Ca2+ influxes through Store Operated Channels (SOCs). When these influxes were blocked by BTP2, a known inhibitor of SOCs, differentiation was abolished. It is worth mentioning that differentiation was not affected by BTP2 in the absence of thapsigargin, suggesting that SOCs channels do not play a major role during normal differentiation. From these experiments, we conclude that human myoblasts can adapt their behavior to generate Ca2+ signals required for differentiation depending on available Ca2+ sources.
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