Charles R. Bader

Cloning of a human ether-a-go-go potassium channel expressed in myoblasts at the onset of fusion

An early sign of human myoblast commitment to fusion is the expression of a non‐inactivating delayed rectifier K+ current, IK(NI), and an associated membrane potential hyperpolarization. We have isolated the full‐length coding region of a human ether‐a‐go‐go K+ channel (h‐eag) from myoblasts undergoing differentiation. The h‐eag gene was localized to chromosome 1q32–41, and is expressed as a ∼9 kb transcript in myogenic cells and in adult brain tissue. Forced expression of h‐eag in undifferentiated myoblasts generates a current with remarkable similarity to IK(NI) indicating that h‐eag constitutes the channel responsible for this current in vivo.

STIM1- and Orai1-dependent Store-operated Calcium Entry Regulates Human Myoblast Differentiation

Our previous work on human myoblasts suggested that a hyperpolarization followed by a rise in [Ca2+]in involving store-operated Ca2+ entry (SOCE) channels induced myoblast differentiation. Advances in the understanding of the SOCE pathway led us to examine more precisely its role in post-natal human myoblast differentiation. We found that SOCE orchestrated by STIM1, the endoplasmic reticulum Ca2+ sensor activating Orai Ca2+ channels, is crucial. Silencing STIM1, Orai1, or Orai3 reduced SOCE amplitude and myoblast differentiation, whereas Orai2 knockdown had no effect. Conversely, overexpression of STIM1 with Orai1 increased SOCE and accelerated myoblast differentiation. STIM1 or Orai1 silencing decreased resting [Ca2+]in and intracellular Ca2+ store content, but correction of these parameters did not rescue myoblast differentiation. Remarkably, SOCE amplitude correlated linearly with the expression of two early markers of myoblast differentiation, MEF2 and myogenin, regardless of the STIM or Orai isoform that was silenced. Unexpectedly, we found that the hyperpolarization also depends on SOCE, placing SOCE upstream of K+ channel activation in the signaling cascade that controls myoblast differentiation. These findings indicate that STIM1 and Orai1 are key molecules for the induction of human myoblast differentiation.

STIM1L is a new actin-binding splice variant involved in fast repetitive Ca2+ release

A newly identified splice variant of STIM1 called STIM1L forms constitutive clusters that interact with actin and Orai1 and allows fast repetitive Ca2+ release.

An ether ‐à‐go‐go K+ current, Ih‐eag, contributes to the hyperpolarization of human fusion‐competent myoblasts

1 Two early signs of human myoblast commitment to fusion are membrane potential hyperpolarization and concomitant expression of a non‐inactivating delayed rectifier K+ current, IK(NI). This current closely resembles the outward K+ current elicited by rat ether‐à‐go‐go (r‐eag) channels in its range of potential for activation and unitary conductance. 2 It is shown that activation kinetics of IK(NI), like those of r‐eag, depend on holding potential and on [Mg2+]o, and that IK(NI), like r‐eag, is reversibly inhibited by a rise in [Ca2+]. 3 Forced expression of an isolated human ether‐à‐go‐go K+ channel (h‐eag) cDNA in undifferentiated myoblasts generates single‐channel and whole‐cell currents with remarkable similarity to IK(NI). 4 h‐eag current (Ih‐eag) is reversibly inhibited by a rise in [Ca2+]i, and the activation kinetics depend on holding potential and [Mg2+]o. 5 Forced expression of h‐eag hyperpolarizes undifferentiated myoblasts from −9 to −50 mV, the threshold for the activation of both Ih‐eag and IK(NI). Similarly, the higher the density of IK(NI), the more hyperpolarized the resting potential of fusion‐competent myoblasts. 6 It is concluded that h‐eag constitutes the channel underlying IK(NI) and that it contributes to the hyperpolarization of fusion‐competent myoblasts. To our knowledge, this is the first demonstration of a physiological role for a mammalian eag K+ channel.

Role of an inward rectifier K+ current and of hyperpolarization in human myoblast fusion.

1 The role of K+ channels and membrane potential in myoblast fusion was evaluated by examining resting membrane potential and timing of expression of K+ currents at three stages of differentiation of human myogenic cells: undifferentiated myoblasts, fusion‐competent myoblasts (FCMBs), and freshly formed myotubes. 2 Two K+ currents contribute to a hyperpolarization of myoblasts prior to fusion: IK(NI), a non‐inactivating delayed rectifier, and IK(IR), an inward rectifier. 3 I K(NI) density is low in undifferentiated myoblasts, increases in FCMBs and declines in myotubes. On the other hand, IK(IR) is expressed in 28 % of the FCMBs and in all myotubes. 4 I K(IR) is reversibly blocked by Ba2+ or Cs+. 5 Cells expressing IK(IR) have resting membrane potentials of −65 mV. A block by Ba2+ or Cs+ induces a depolarization to a voltage determined by IK(NI) (‐32 mV). 6 Cs+ and Ba2+ ions reduce myoblast fusion. 7 It is hypothesized that the IK(IR)‐mediated hyperpolarization allows FCMBs to recruit Na+, K+ and T‐type Ca2+ channels which are present in these cells and would otherwise be inactivated. FCMBs, rendered thereby capable of firing action potentials, could amplify depolarizing signals and may accelerate fusion.

Membrane Hyperpolarization Triggers Myogenin and Myocyte Enhancer Factor-2 Expression during Human Myoblast Differentiation

It is widely thought that myogenin is one of the earliest detectable markers of skeletal muscle differentiation. Here we show that, during human myoblast differentiation, an inward rectifier K+ channel (Kir2.1) and its associated hyperpolarization trigger expression and activity of the myogenic transcription factors, myogenin and myocyte enhancer factor-2 (MEF2). Furthermore, Kir2.1 current precedes and is required for the developmental increase in expression/activity of myogenin and MEF2. Drugs or antisense reducing Kir2.1 current diminished or suppressed fusion as well as expression/activity of myogenin and MEF2. In contrast, LY294002, an inhibitor of phosphatidylinositol 3-kinase (a pathway controlling initiation of the myogenic program) that inhibited both myogenin/MEF2 expression and fusion, did not affect Kir2.1 current. This non-blockade by LY294002 indicates that Kir2.1 acts upstream of myogenin and MEF2. We propose that Kir2.1 channel activation is a required key early event that initiates myogenesis by turning on myogenin and MEF2 transcription factors via a hyperpolarization-activated Ca2+-dependent pathway.

Mibefradil (Ro 40-5967) inhibits several Ca2+ and K+ currents in human fusion-competent myoblasts

The effect of mibefradil (Ro 40–5967), an inhibitor of T‐type Ca2+ current (ICa(T)), on myoblast fusion and on several voltage‐gated currents expressed by fusion‐competent myoblasts was examined. At a concentration of 5 μM, mibefradil decreases myoblast fusion by 57%. At this concentration, the peak amplitudes of ICa(T) and L‐type Ca2+ current (ICa(L)) measured in fusion‐competent myoblasts are reduced by 95 and 80%, respectively. The IC50 of mibefradil for ICa(T) and ICa(L) are 0.7 and 2 μM, respectively. At low concentrations, mibefradil increased the amplitude of ICa(L) with respect to control. Mibefradil blocked three voltage‐gated K+ currents expressed by human fusion‐competent myoblasts: a delayed rectifier K+ current, an ether‐à‐go‐go K+ current, and an inward rectifier K+ current, with a respective IC50 of 0.3, 0.7 and 5.6 μM. It is concluded that mibefradil can interfere with myoblast fusion, a mechanism fundamental to muscle growth and repair, and that the interpretation of the effect of mibefradil in a given system should take into account the action of this drug on ionic currents other than Ca2+ currents.

The calcineurin pathway links hyperpolarization (Kir2.1)-induced Ca2+ signals to human myoblast differentiation and fusion

In human myoblasts triggered to differentiate, a hyperpolarization, resulting from K+ channel (Kir2.1) activation, allows the generation of an intracellular Ca2+ signal. This signal induces an increase in expression/activity of two key transcription factors of the differentiation process, myogenin and MEF2. Blocking hyperpolarization inhibits myoblast differentiation. The link between hyperpolarization-induced Ca2+ signals and the four main regulatory pathways involved in myoblast differentiation was the object of this study. Of the calcineurin, p38-MAPK, PI3K and CaMK pathways, only the calcineurin pathway was inhibited when Kir2.1-linked hyperpolarization was blocked. The CaMK pathway, although Ca2+ dependent, is unaffected by changes in membrane potential or block of Kir2.1 channels. Concerning the p38-MAPK and PI3K pathways, their activity is present already in proliferating myoblasts and they are unaffected by hyperpolarization or Kir2.1 channel block. We conclude that the Kir2.1-induced hyperpolarization triggers human myoblast differentiation via the activation of the calcineurin pathway, which, in turn, induces expression/activity of myogenin and MEF2.

Human Muscle Economy Myoblast Differentiation and Excitation-Contraction Coupling Use the Same Molecular Partners, STIM1 and STIM2

Our recent work identified store-operated Ca2+ entry (SOCE) as the critical Ca2+ source required for the induction of human myoblast differentiation (Darbellay, B., Arnaudeau, S., König, S., Jousset, H., Bader, C., Demaurex, N., and Bernheim, L. (2009) J. Biol. Chem. 284, 5370–5380). The present work indicates that STIM2 silencing, similar to STIM1 silencing, reduces myoblast SOCE amplitude and differentiation. Because myoblasts in culture can be induced to differentiate into myotubes, which spontaneously contract in culture, we used the same molecular tools to explore whether the Ca2+ mechanism of excitation-contraction coupling also relies on STIM1 and STIM2. Live cell imaging of early differentiating myoblasts revealed a characteristic clustering of activated STIM1 and STIM2 during the first few hours of differentiation. Thapsigargin-induced depletion of endoplasmic reticulum Ca2+ content caused STIM1 and STIM2 redistribution into clusters, and co-localization of both STIM proteins. Interaction of STIM1 and STIM2 was revealed by a rapid increase in fluorescence resonance energy transfer between CFP-STIM1 and YFP-STIM2 after SOCE activation and confirmed by co-immunoprecipitation of endogenous STIM1 and STIM2. Although both STIM proteins clearly contribute to SOCE and are required during the differentiation process, STIM1 and STIM2 are functionally largely redundant as overexpression of either STIM1 or STIM2 corrected most of the impact of STIM2 or STIM1 silencing on SOCE and differentiation. With respect to excitation-contraction, we observed that human myotubes rely also on STIM1 and STIM2 to refill their endoplasmic reticulum Ca2+-content during repeated KCl-induced Ca2+ releases. This indicates that STIM2 is a necessary partner of STIM1 for excitation-contraction coupling. Thus, both STIM proteins are required and interact to control SOCE during human myoblast differentiation and human myotube excitation-contraction coupling.

Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured human myoblasts.

1. Fusion of myogenic cells is important for muscle growth and repair. The aim of this study was to examine the possible involvement of nicotinic acetylcholine receptors (nAChR) in the fusion process of myoblasts derived from postnatal human satellite cells. 2. Acetylcholine‐activated currents (ACh currents) were characterized in pure preparations of freshly isolated satellite cells, proliferating myoblasts, myoblasts triggered to fuse and myotubes, using whole‐cell and single‐channel voltage clamp recordings. Also, the effect of cholinergic agonists on myoblast fusion was tested. 3. No nAChR were observed in freshly isolated satellite cells. nAChR were first observed in proliferating myoblasts, but ACh current densities increased markedly only just before fusion. At that time most mononucleated myoblasts had ACh current densities similar to those of myotubes. ACh channels had similar properties at all stages of myoblast maturation. 4. The fraction of myoblasts that did not fuse under fusion‐promoting conditions had no ACh current and thus resembled freshly isolated satellite cells. 5. The rate of myoblast fusion was increased by carbachol, an effect antagonized by alpha‐bungarotoxin, curare and decamethonium, but not by atropine, indicating that nAChR were involved. Even though a prolonged exposure to carbachol led to desensitization, a residual ACh current persisted after several days of exposure to the nicotinic agonist. 6. Our observations suggest that nAChR play a role in myoblast fusion and that part of this role is mediated by the flow of ions through open ACh channels.