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

Cytosolic Ca(2+) signals encoded by repetitive Ca(2+) releases rely on two processes to refill Ca(2+) stores: Ca(2+) reuptake from the cytosol and activation of a Ca(2+) influx via store-operated Ca(2+) entry (SOCE). However, SOCE activation is a slow process. It is delayed by >30 s after store depletion because stromal interaction molecule 1 (STIM1), the Ca(2+) sensor of the intracellular stores, must form clusters and migrate to the membrane before being able to open Orai1, the plasma membrane Ca(2+) channel. In this paper, we identify a new protein, STIM1L, that colocalizes with Orai1 Ca(2+) channels and interacts with actin to form permanent clusters. This property allowed the immediate activation of SOCE, a characteristic required for generating repetitive Ca(2+) signals with frequencies within seconds such as those frequently observed in excitable cells. STIM1L was expressed in several mammalian tissues, suggesting that many cell types rely on this Ca(2+) sensor for their Ca(2+) homeostasis and intracellular signaling.

Human muscle economy myoblast differentiation and excitation-contraction coupling use the same molecular partners, STIM1 and STIM2.

Our recent work identified store-operated Ca(2+) entry (SOCE) as the critical Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-content during repeated KCl-induced Ca(2+) 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.

Myogenic precursor cell transplantation in pigs: a step towards a clinical use for muscle regeneration?

It is most probable that, in a near future, myogenic precursor cell transplants will have clinical applications in domains as different as orthopaedics, endocrinology, management of heart infarct, and therapies of muscle diseases. We have proposed to introduce the use of myogenic precursor cell transplantation in patients, after preliminary tests in a large animal model, the pig. Our initial effort was centred on the domain of orthopaedics. Muscle damages are frequent complications of traumas and sport accidents with serious consequences both in terms of disabilities and health economics. Often these lesions heal very poorly. A number of growth factors seemed successful as healing agents but they are difficult to deliver clinically. The goal was to use ex vivo somatic gene therapy with myogenic precursor cells modified to secrete growth factors with the aim of improving muscle healing in patients and of demonstrating the potential of this technology. To do so, we used a suitable large animal model, the pig, for exploring myogenic precursor cell transplantation strategies that could be used in patients.

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.

Calcium sources used by post-natal human myoblasts during initial differentiation.

Increases in cytoplasmic Ca(2+) are crucial for inducing the initial steps of myoblast differentiation that ultimately lead to fusion; yet the mechanisms that produce this elevated Ca(2+) have not been fully resolved. For example, it is still unclear whether the increase comes exclusively from membrane Ca(2+) influx or also from Ca(2+) release from internal stores. To address this, we investigated early differentiation of myoblast clones each derived from single post-natal human satellite cells. Initial differentiation was assayed by immunostaining myonuclei for the transcription factor MEF2. When Ca(2+) influx was eliminated by using low external Ca(2+) media, we found that approximately half the clones could still differentiate. Of the clones that required influx of external Ca(2+), most clones used T-type Ca(2+) channels, but others used store-operated channels as influx-generating mechanisms. On the other hand, clones that differentiated in low external Ca(2+) relied on Ca(2+) release from internal stores through IP(3) receptors. Interestingly, by following clones over time, we observed that some switched their preferred Ca(2+) source: clones that initially used calcium release from internal stores to differentiate later required Ca(2+) influx and inversely. In conclusion, we show that human myoblasts can use three alternative mechanisms to increase cytoplasmic Ca(2+) at the onset of the differentiation process: influx through T-types Ca(2+) channels, influx through store operated channels and release from internal stores through IP(3) receptors. In addition, we suggest that, probably because Ca(2+) elevation is essential during initial differentiation, myoblasts may be able to select between these alternate Ca(2+) pathways.

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 Ca(2+)-dependent pathway.

Acceleration of human myoblast fusion by depolarization: graded Ca2+ signals involved.

We have previously shown that human myoblasts do not fuse when their voltage fails to reach the domain of a window T-type Ca(2+) current. We demonstrate, by changing the voltage in the window domain, that the Ca(2+) signal initiating fusion is not of the all-or-none type, but can be graded and is interpreted as such by the differentiation program. This was carried out by exploiting the properties of human ether-à-go-go related gene K(+) channels that we found to be expressed in human myoblasts. Methanesulfonanilide class III antiarrhythmic agents or antisense-RNA vectors were used to suppress completely ether-à-go-go related gene current. Both procedures induced a reproducible depolarization from -74 to -64 mV, precisely in the window domain where the T-type Ca(2+) current increases with voltage. This 10 mV depolarization raised the cytoplasmic free Ca(2+) concentration, and triggered a tenfold acceleration of myoblast fusion. Our results suggest that any mechanism able to modulate intracellular Ca(2+) concentration could affect the rate of myoblast fusion.

Presence of functional oxytocin receptors in cultured human myoblasts.

In the present report, we provide for the first time evidence that functional oxytocin receptors (OTRs) are present in human myoblasts obtained from clonal cultures of postnatal satellite cells. First, binding studies performed with a non selective vasopressin (AVP) and oxytocin (OT) radioligand indicated the presence of a single class of binding sites. Second, OTR mRNA was detected by RT-PCR analysis whereas transcripts for AVP V(1a), V(1b) or V(2) receptors (V(1a)R, V(1b)R and V(2)R respectively) were not detected. Third, the presence of functional OTRs was evidenced by showing that agonist substances having a high affinity for the human OTR, namely OT, AVP and [Thr(4)Gly(7)]OT, increased the rate of myoblasts fusion and myotubes formation in the cultures, whereas F180, a V(1a)R selective agonist, and dDAVP, a V(2)R agonist had no significant effect on the fusion process. In addition, we show by RT-PCR and immunocytochemistry that the OT gene is expressed in cultured myoblasts. Taken together, our data suggest that OT may act as a paracrine/autocrine agent that stimulates the fusion of human myoblasts in vitro. In vivo, OT may be involved in the differentiation of human skeletal muscle during postnatal growth, and possibly its regeneration following injury.

Human myoblast differentiation: Ca(2+) channels are activated by K(+) channels.

In a paradigm of cellular differentiation, human myoblast fusion, we investigated how a Ca(2+) influx, indispensable for fusion, is triggered. We show how newly expressed Kir2.1 K(+) channels, via their hyperpolarizing effect on the membrane potential, generate a window Ca(2+) current (mediated by alpha 1H T-type Ca(2+) channels), which causes intracellular Ca(2+) to rise.