Résumé :
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Our work is based on human primary myoblast cultures derived from single satellite cells. In human, as in other species, it is well established that myoblast differentiation is mainly controlled by two families of transcription factors, the Myogenic Regulatory Factors (including MyoD and myogenin) and the MEF-2 (A-D) family. Initiation of myogenesis is controlled by pre-existing transcriptions factors like MyoD. At later stages of muscle differentiation, myogenin is expressed and together with MEF- 2D is thought to control the expression of late genes such as MEF-2C and the genes encoding muscle-specific proteins.We have previously shown that, during the differentiation process of human myoblasts, the expression of myogenic transcription factors follows a hyperpolarization of the membrane myoblast resting potential from 40 mV to 70 mV due to Kir2.1 channel activation. Inhibition of this hyperpolarization strongly decreases myoblast differentiation, indicating that the hyperpolarization is a prerequisite for myogenic factor expression. When induced to differentiate, human myoblasts first hyperpolarize and then express myogenin and, slightly later MEF2 factors. In the present study, we show that inhibition of membrane hyperpolarization with 10 mM cesium does not modify myogenin expression but strongly decreases MEF2 expression. At the transcriptional level, MEF-2A, -2C and -2D are upregulated during myoblast differentiation. However, at the protein level MEF-2D is the only isoform to be already detected in proliferating myoblasts and its up-regulation is mainly restricted to nuclei incorporated into myotubes. Once expressed, MEF2A or MEF2C proteins are primarily detected in nuclei present in myotubes. When hyperpolarization is inhibited by 10 mM cesium, expression of MEF-2A and -2C is strongly inhibited. Surprisingly, neither MEF-2D expression, nor MyoD, myogenin or myf5 expression are modified in presence of 10 mM cesium.Our results suggest that the Kir2.1-linked hyperpolarization (and the related calcium signals) triggers MEF-2A/-2C expression and then late muscle-specific genes, while myogenin expression is controlled by another pathway.We propose that early myoblast hyperpolarization allows activation of the pre-existing factors MyoD and MEF-2D that trigger MEF-2A 2C expression. First results show that MEF-2D is differentially regulated at the post-translational level during early and late stages of myogenesis.
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