Project

Discipline

human muscle; myoblast differentiation; ionic channels; electrophysiology; myogenic transcription factors; calcium; SOCE; cameleon indicators

Lead
Lay summary
The general goal of our lab is to study human skeletal muscle regeneration. Muscle regeneration is a paradigm of a differentiation process as it involves myogenic differentiation factors, it is controlled by extracellular growth factors and mediators, and it works through the activation of multiple intracellular signaling pathways. Our starting material is the satellite cell, the myogenic stem cell of skeletal muscle. Upon muscle injury, satellite cells proliferate as myoblasts and, after a process of differentiation, fuse together to generate new muscle fibers. Our objective is to understand the interplay between ionic channels, resting membrane potential, intracellular calcium, and various enzymes or myogenic regulatory factors activated during the differentiation process of myoblasts. We discovered that a membrane hyperpolarization is not only among the earliest steps in myoblast differentiation but is also required for the differentiation process to take place. This membrane hyperpolarization results from an up regulation of two potassium channels: ether à-go-go and Kir2.1 inward rectifying potassium channels. We found that the purpose of the differentiation-linked hyperpolarization is to generate calcium influxes mainly via store-operated Orai1 channels and voltage-gated T-type calcium channels. These calcium influxes, in turn, via the CaMK and calcineurin pathways, activate the myogenic transcription factors myogenin and MEF2, two transcription factors crucial for myoblast differentiation.

Direct link to Lay Summary

Last update: 21.02.2013

Number	Title	Start	Funding scheme

With the long-term goal of improving the survival of myoblasts injected for the treatment of muscle as well as non-muscle-related disorders, our group aims at unraveling the early steps of human myoblast differentiation that leads to myoblast fusion. Our previous work demonstrated that hyperpolarization of the membrane potential was one of the firsts events to occur. More recently, we discovered that a dephosphorylation at tyrosine 242 opens Kir2.1 inward rectifying K+ channels and thereby induces the hyperpolarization of myoblasts. The resulting increased driving force for Ca2+ caused by hyperpolarization promotes Ca2+ influx through Ca2+ channels. Another recent observation of our group is that several types of Ca2+ channels can provide the calcium signal required for differentiation, including store operated Ca2+ entry (SOCE) channels. Interestingly, we found that siRNAs-mediated silencing of the key players of SOCE channels reduced SOCE and impaired myoblast differentiation. On the contrary, overexpression of the key players of SOCE amplified SOCE and stimulated myoblast differentiation. Also, our electrophysiological and molecular biology data suggest that two Ca2+ signals are required to trigger human myoblast differentiation. These recent findings have lead us to review our working hypothesis for the early steps of myoblast differentiation and to propose the three following general objectives:1. Extend our understanding of the relationships between hyperpolarization, Ca2+ dependent pathways, and expression of MEF2 transcription factors. We plan to study the early steps of myoblast differentiation that control the expression of myogenin and MEF2 transcription factors and which are linked to ionic channel activity.2. Confirm that two Ca2+ signals are required to trigger human myoblast differentiation. We aim at identifying these two Ca2+ signals, and examine their correlation with CaMK and calcineurin activation. We shall follow cytoplasmic Ca2+ changes with various targeted dyes and examine whether these changes are primarily due to Ca2+ influxes through membrane channels or Ca2+ release from internal stores.3. Elucidate the pathways controlling the activation of K+ channels at the onset of myoblast differentiation. We intend to identify the kinases and phosphatases involved in the activation of Kir2.1 channels at the beginning of the differentiation process and evaluate their possible modulation by SOCE.These 3 projects should improve our understanding of human myoblasts differentiation. In addition, as all cells express and probably rely on ionic channels, our work on myoblasts might shed light on mechanisms involved in cellular differentiation in general.