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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.

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.

Myoblast differentiation is essential to skeletal muscle formation and repair. The earliest detectable event leading to human myoblast differentiation is an upregulation of Kir2.1 channel activity, which causes a negative shift (hyperpolarization) of the resting potential of myoblasts. After exploring various mechanisms, we found that this upregulation of Kir2.1 was due to dephosphorylation of the channel itself. Application of genistein, a tyrosine kinase inhibitor, increased Kir2.1 activity and triggered the differentiation process, whereas application of bpV(Phen), a tyrosine phosphatase inhibitor, had the opposite effects. We could show that increased Kir2.1 activity requires dephosphorylation of tyrosine 242; replacing this tyrosine in Kir2.1 by a phenylalanine abolished inhibition by bpV(Phen). Finally, we found that the level of tyrosine phosphorylation in endogenous Kir2.1 channels is considerably reduced during differentiation when compared with proliferation. We propose that Kir2.1 channels are already present at the membrane of proliferating, undifferentiated human myoblasts but in a silent state, and that Kir2.1 tyrosine 242 dephosphorylation triggers differentiation.

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.

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.

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.

In inclusion body myositis (IBM), there is muscular amyloidogenesis and inflammation. A related disorder is due to alterations in the ubiquitin pathway involving the valsolin-containing protein leading to IBM, dementia and Paget's disease. Alteration in the dystrophin glycoprotein complex leads to several muscular dystrophies (MD), and the pathogenesis of dystrophin related MD as well as certain limb girdle MD are discussed. Therapeutic strategies involving inhibition of proteolytic cascades as well as inhibition of a negative regulator of muscle growth (myostatin) are briefly introduced. Finally, molecular aspects of the most common form of adult myopathy, myotonic MD, are discussed. This disease is caused by an aberrant splicing mechanism and interference thereof may be useful in designing therapeutic strategies.

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.

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.

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.

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.

Myoblast fusion is essential to skeletal muscle development and repair. We have demonstrated previously that human myoblasts hyperpolarize, before fusion, through the sequential expression of two K+ channels: an ether-à-go-go and an inward rectifier. This hyperpolarization is a prerequisite for fusion, as it sets the resting membrane potential in a range at which Ca2+ can enter myoblasts and thereby trigger fusion via a window current through alpha1H T channels.

Gene transfer of human primary myoblasts with various non-viral methods has been hampered by low yield of transfection. We report here an efficient, simple and reproducible non-viral DNA-mediated gene transfer procedure for transfecting human myoblasts. We found that electroporation promotes a highly efficient DNA uptake by human primary cultures of myogenic cells. Under optimal conditions, 60-70% of human myoblasts transfected with the enhanced green fluorescent gene expressed the enhanced green fluorescent protein. Electroporated myoblasts behaved normally as judged by their ability to synthesize and express developmentally regulated proteins and to undergo terminal differentiation, i.e. to fuse and form myotubes. We showed, in addition, that a subpopulation of cultured human myoblasts with self-renewing properties and equivalent to native muscle satellite cells were as efficiently transfected by electroporation as proliferating myoblasts. Thus, the development of gene therapies based on the engineering and transplantation of human myoblasts may greatly benefit from gene transfer by electroporation.

Mechanisms underlying Ca(2+) signaling during human myoblast terminal differentiation were studied using cell cultures. We found that T-type Ca(2+) channels (T-channels) are expressed in myoblasts just before fusion. Their inhibition by amiloride or Ni(2+) suppresses fusion and prevents an intracellular Ca(2+) concentration increase normally observed at the onset of fusion. The use of antisense oligonucleotides indicates that the functional T-channels are formed by alpha1H subunits. At hyperpolarized potentials, these channels allow a window current sufficient to increase [Ca(2+)](i). As hyperpolarization is a prerequisite to myoblast fusion, we conclude that the Ca(2+) signal required for fusion is produced when the resting potential enters the T-channel window. A similar mechanism could operate in other cell types of which differentiation implicates membrane hyperpolarization.

1. The effect of mibefradil (Ro 40-5967), an inhibitor of T-type Ca2+ current (I(Ca)(T)), on myoblast fusion and on several voltage-gated currents expressed by fusion-competent myoblasts was examined. 2. At a concentration of 5 microM, mibefradil decreases myoblast fusion by 57%. At this concentration, the peak amplitudes of I(Ca)(T) and L-type Ca2+ current (I(Ca)(L)) measured in fusion-competent myoblasts are reduced by 95 and 80%, respectively. The IC50 of mibefradil for I(Ca)(T) and I(Ca)(L) are 0.7 and 2 microM, respectively. 3. At low concentrations, mibefradil increased the amplitude of I(Ca)(L) with respect to control. 4. 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 microM. 5. 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.

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+]i. 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.

An early sign of human myoblast commitment to fusion is the expression of a non-inactivating delayed rectifier K+ current, I(K(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 approximately 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 I(K(NI)) indicating that h-eag constitutes the channel responsible for this current in vivo.

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. IK(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. IK(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.

We have evaluated needle muscle biopsies in 220 patients with various neuromuscular disorders, using a method developed previously at Tuft's University. The method uses a 14-gauge needle propelled by an automatic device. An average of 3.5 samplings were taken per patient. Muscle samples were used for histological and molecular genetic analysis, and for the isolation of muscle satellite cells for in vitro cultures. The biopsy is well tolerated by the patients who never declined multiple samplings. Complications were few and minor, with no sequelae. In most cases the small size of the muscle specimen (ca. 15 mg per sampling) was sufficient to perform the various procedures and to yield a diagnosis. Specimens were considered insufficient for histological results in 9 patients (4%), due to technical artifacts or insufficient material. We now routinely use this method, which has several advantages over the surgical technique for most muscle biopsies.

1. Using the patch-clamp technique, a new non-inactivating voltage-gated potassium current, IK(ni), was studied in cultured fusion-competent human myoblasts. 2. IK(ni) is activated at voltages above -50 mV and its conductance reaches its maximum around +50 mV. Once activated, the current remains at a steady level for minutes. 3. Reversal potential measurements at various extracellular potassium concentrations indicate that potassium ions are the major charge carriers of IK(ni). 4. IK(ni) is insensitive to potassium channel blockers such as charybdotoxin, dendrotoxins, mast cell degranulating (MCD) peptide, 4-aminopyridine (4-AP), 3,4-diaminopyridine (3,4-DAP) and apamin, but can be blocked by high concentrations of TEA and by Ba2+. 5. A potassium channel of small conductance (8.4 pS at +40 mV) with potential dependence and pharmacological properties corresponding to those of IK(ni) in whole-cell recording is described. 6. IK(ni) participates in the control of the resting potential of fusion-competent myoblasts, suggesting that it may play a key role in the process of myoblast fusion.

It has long been known that nicotinic acetycholine receptors (nAChRs) are present in muscle fibres not only at the end plate region but also at the myotendinous junction (MTJ). Their function at the MTJ, however, is yet unknown. Recent experiments in our laboratory lead us to suggest that nAChRs at this site might be involved in muscle repair. MTJ is subject to high mechanical stress and therefore is easily damaged. We found in pure cultures of human myogenic cells that (1) the density of nAChRs in myoblasts increases markedly just before cell fusion, (2) the fusion of human myoblasts is accelerated by the presence of a cholinergic agonist acting on nAChRs and (3) human myoblasts and myotubes spontaneously release an ACh-like compound. Based on these observations we propose that in damaged muscles the nAChRs at the MTJ and those of myogenic cells are activated by the ACh-like compound these cells release. This leads to fusion of myogenic cells with damaged muscle fibres and hence promotes repair.

We demonstrate that self-renewing myoblasts can be identified in the progeny of single human muscle satellite cells (HMSC) in culture. We show, using cytoskeletal proteins and cell size as markers, that self-renewing myoblasts are phenotypically different from other myoblasts, but similar to native HMSC. Native desmin-positive HMSC, cultured as single cells, yielded two major populations of myoblasts, alpha-sarcomeric (alpha-SR)-actin-positive myoblasts and desmin-positive myoblasts. In appropriate culture conditions, alpha-SR-actin-positive myoblasts fused into myotubes, whereas a population of desmin-positive non-fusing myoblasts (NFMB) persisted for weeks among the myotubes. Upon isolation from myotubes, some of the NFMB resumed proliferation and their progeny included fusing and non-fusing myoblasts, with the same cytoskeletal phenotypes as the progeny of native HMSC. This self-renewal cycle could be repeated, yielding four cohorts of myoblasts. The yield of self-renewing cells appeared to decrease with the number of cycles. These results suggest that stem cells are present among NFMB. Moreover, we find that these presumptive stem cells are already segregated during myoblast proliferation. They are small, phenotypically similar to native HMSC, and do not divide unless they are isolated from their sister progeny and cultured alone. Enriched preparations of cells with stem cell-like properties can be obtained from proliferating myoblasts by flow cytometry on the basis of size and nucleocytoplasmic ratio.

1. Exogenously applied acetylcholine (ACh) is a modulator of human myoblast fusion. Using a chemiluminescent method, we examined whether an endogenous ACh-like compound (ACh-lc) was present in, and released by, pure human myogenic cells. 2. Single, freshly isolated satellite cells and proliferating myoblasts contained 15 and 0.5 fmol ACh-lc, respectively. Cultured myotubes contained ACh-lc as well. Also, ACh-like immunoreactivity was detected in all myogenic cells. 3. Part of the ACh-lc was synthesized by choline acetyltransferase (ChAT), as indicated by the reduction of ACh-lc content when bromoACh was present in the culture medium, and by direct measurements of ChAT activity. Also, ChAT-like immunoreactivity was observed in all myogenic cells. 4. Myoblasts and myotubes released ACh-lc spontaneously by a partially Ca(2+)-dependent mechanism. 5. The application by microperfusion of medium conditioned beforehand by myoblasts (thus presumably containing ACh-lc) onto a voltage-clamped myotube induced inward currents resembling ACh-induced currents in their kinetics, reversal potential, and sensitivity to nicotinic antagonists. 6. In vitro, the spontaneously released ACh-lc promoted myoblast fusion but only in the presence of an anticholinesterase. 7. Our observations indicate that human myogenic cells synthesize and release an ACh-lc and thereby promote the fusion process that occurs in muscle during growth or regeneration.

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.

We examined whether freshly isolated (native) human muscle satellite cells (HMSC), as well as their proliferating clonal progenies, were heterogeneous. We studied the expression of the cytoskeletal proteins, desmin (DSM), alpha-sarcomeric and alpha-smooth muscle actins (alpha-SR actin, alpha-SM actin), three markers that may be expressed prior to the fusion process. We found that native HMSC constituted a homogeneous population of cells expressing desmin and giving rise to similar clones in vitro. The clonal progeny of HMSC was heterogeneous, including several subpopulations of myoblasts with different cytoskeletal phenotypes, commitment states and fusion abilities. A major subpopulation that expressed both alpha-sarcomeric actin and desmin during the proliferative stage corresponded to a "predifferentiated" population of myoblasts, committed to fusion. Another subpopulation, expressing exclusively desmin, and phenotypically similar to native HMSC, failed to fuse under fusion-promoting conditions and could represent a new generation of HMSC born in culture.

Using the whole-cell recording patch clamp technique in clonal cultures of human muscle satellite cells (SC), we studied a voltage-gated potassium current analogous to the delayed rectifier current (IKdr) described in adult human skeletal muscle. This current was absent in proliferating SC cultured in a growth medium containing 15% serum, except when the SC approached the end of their replicative life (between 77 and 124 days in culture); at that time, approximately 50% of the SC possessed IKdr. In contrast, IKdr was expressed within less than 4 days in approximately 70% of the SC cultured in a serum-free medium (SFM) and within 24 h in differentiating medium. We believe that IKdr may be a characteristic feature of fusion-component SC and that it may be involved in the fusion process for the following reasons: 1) after the transfer in differentiating medium, cultures of SC in which the expression of IKdr was previously promoted by exposure to SFM were found to fuse immediately, without the initial 24 h lag time observed in control sister cultures; 2) in the latter "naive" SC, IKdr was expressed during the first day in differentiating medium, before SC began to fuse; 3) most of the SC that did not fuse even after weeks of exposure to differentiating medium did not express IKdr; 4) TEA, at a concentration of 3 mM, reduces the amplitude of IKdr by 55% and the fusion index by 55-67%.

1. Human muscle satellite cells (SC) were studied either immediately after dissociation of muscle biopsies or later, as they proliferated in culture. A purification procedure combined with clonal cultures ensured that electrophysiological recordings were done in myogenic cells. Hoechst staining for the DNA attested that cells were mononucleated. 2. The goals of this study were to examine (i) whether the electrophysiological properties of freshly isolated SC resembled those of SC that proliferated in culture for several weeks, (ii) whether freezing and thawing affected these properties, and (iii) whether SC constituted a homogeneous population. 3. We found that there were only subtle differences between the electrophysiological results obtained in freshly isolated SC and in proliferating SC with or without previous freezing and thawing. Most SC expressed two voltage-gated currents, a TTX-resistant Na+ current and a calcium-activated potassium current (IK, Ca). 4. The level of expression of the Na+ current and of IK, Ca was affected in a different way by cellular proliferation; the normalized Na+ conductance (pS pF-1) of proliferating cells resembled that of freshly isolated SC, whereas the IK, Ca conductance increased 10 times. The analysis of the amplitude distributions of the Na+ current and of IK, Ca in the various SC preparations suggested that there was only one class of SC.

1. A voltage-dependent proton current, IH, was studied in cultured myotubes obtained from biopsies of human muscle, using whole-cell recording with the patch-clamp technique. 2. With a pHo of 8.0 and a calculated pHi of 6.3, IH was activated at voltages more depolarized than -50 mV and its conductance reached its maximum value at voltages more depolarized than +10 mV. 3. Studies of the reversal potential of IH during substitution of K+, Na+, Ca2+, Cl-, Cs+ and H+ in the extracellular solution indicated that protons were the major charge carriers of IH. 4. IH was also activated during a voltage step to +22 mV with a pHo of 7.3 and a calculated pHi of 7.3. 5. Acidification of the extracellular solution led to a shift towards depolarized voltages of the conductance-voltage relationship. 6. Stationary noise analysis of IH suggested that the elementary event underlying IH was very small with a conductance of less than 0.09 pS. 7. Extracellular application of various divalent cations blocked IH. The block by divalent cations was voltage dependent, being more efficient at hyperpolarized than at depolarized voltages. For Cd2+, the Michaelis-Menten constant (Km) for the block was 0.6 microM at -28 mV and 10.4 microM at +12 mV. 8. Ca2+ was a less efficient blocker than Cd2+ but could block IH at physiological concentrations (the Km values for the block were 0.9 mM at -38 mV and 7.3 mM at -8 mV). 9. The voltage-dependent properties of IH and its ability to be affected by pH and Ca2+ suggest that IH might be used by skeletal muscle cells to extrude protons during action potentials. 10. A model of IH activation suggests that under extreme conditions, the conductance of IH can reach 40% of its maximum value after less than ten action potentials.

A voltage-gated proton current, IH, was studied with the whole-cell patch-clamp technique in human myotubes obtained from biopsies of human muscle. Studies of the reversal potential of IH during substitution of K+, Na+, Ca2+, Cl-, Cs+, and H+ in the extracellular solution indicated that protons were the major charge carriers of IH. This current is similar in many respects, but not identical, to the proton currents already described in other cell types. IH is activated by depolarization and it can be affected by extracellular pH. IH can be blocked by external divalent cations including Ca2+. This block is voltage-dependent, being more efficient at hyperpolarized than at depolarized voltages. The voltage-dependent properties of IH and its ability to be affected by pH and extracellular Ca2+ suggest that IH might be used by muscle cells to extrude protons during action potentials.

To purify satellite cells directly from human muscle biopsies, we have developed a method based on size separation of dissociated cells by flow cytometry. Immediately after tryptic dissociation of human muscle biopsies and elimination of erythrocytes, microscopic observation and flow cytometry analysis of cell suspensions revealed two populations of cells differing in size and nucleocytoplasmic ratio. Clonal cultures of these two cell types with a manual procedure demonstrated that only the small cells were myogenic satellite cells. Flow cytometry-sorting and analysis of the small cell population showed that (1) all sorted cells contained desmin immediately after dissociation and plating; (2) more than 98% of the cells expressed the 5.1.H11 epitope after 2 weeks of proliferation in culture; and (3) 90% of the sorted cells were able to form myotubes when cultivated at low density or in clonal cultures. Thus, human muscle satellite cells can be directly purified from human muscle samples using flow cytometry.

1. The developmental expression of an inwardly rectifying current activated by membrane hyperpolarization (Ih) and of a tetrodotoxin (TTX)-resistant Na+ current (INa(TR)) was studied using freshly dissociated ganglionic quail neurones of various embryonic ages. This work was carried out on parasympathetic (ciliary) and sensory (trigeminal and dorsal root) ganglion neurones with the whole-cell configuration of the patch-clamp technique. 2. In sensory and parasympathetic neurones, Ih was activated at potentials more negative than -60 mV and displayed strong inward rectification. No sign of time- or voltage-dependent inactivation was apparent. Ih was carried by both Na+ and K+ ions and was selectively and reversibly blocked by extracellular Cs+. 3. During the development of sensory neurones, Ih was observed for the first time between embryonic day 10 (E10) and E11 and the percentage of neurones expressing the current increased subsequently, reaching a plateau level of about 80% at E14. In the parasympathetic neurones of the ciliary ganglion, Ih was already detected at E10 and the percentage of neurones possessing the current increased until E16, a stage at which all neurones were found to express Ih. 4. In the presence of TTX (1 microM), an inward Na+ current, INa(TR), was recorded in sensory neurones after E12. This current was activated at potentials more depolarized than -30 mV and its amplitude was maximal at +5 mV. INa(TR) showed time- and voltage-dependent inactivation. Half-maximal steady-state inactivation was observed at -40 mV. 5. INa(TR) was observed for the first time after E12 in sensory neurones and the percentage of neurones with INa(TR) increased until E14. Thereafter, 80% of the neurones had the current. In contrast, INa(TR) was never observed in the parasympathetic neurones of the ciliary ganglion during embryonic development. 6. Our results with parasympathetic and sensory neurones suggest that the expression of INa(TR) is linked to the phenotype and not to the embryonic origin of a neurone.

Whole-cell voltage clamp and single-channel recordings were performed on cultured trigeminal ganglion neurons from quail embryos in order to study a sodium-activated potassium current (KNa). When KNa was activated by a step depolarization in voltage clamp, there was a proportionality between KNa and INa at all voltages between the threshold of INa and ENa. Single-channel recordings indicated that KNa could be activated already by 12 mM intracellular sodium and was almost fully activated at 50 mM sodium. 100 mM lithium, 100 mM choline, or 5 microM calcium did not activate KNa. The relationship between the probability for the channel to be open (Po) vs. the sodium concentration and the relationship of KNa open time-distributions vs. the sodium concentration suggest that two to three sodium ions bind cooperatively before KNa channels open. KNa channels were sensitive to depolarization; at 12 mM sodium, a 42-mV depolarization caused an e-fold increase in Po. Under physiological conditions, the conductance of the KNa channel was 50 pS. This conductance increased to 174 pS when the intra- and extracellular potassium concentrations were 75 and 150 mM, respectively.

Intracellular current administration evokes rapid, graded, and bidirectional mechanical responses of isolated outer hair cells from the mammalian inner ear. The cells become shorter in response to depolarizing and longer in response to hyperpolarizing currents in the synaptic end of the cell. The cells respond with either an increase or decrease in length to transcellular alternating current stimulation. The direction of the movement with transcellular stimuli appears to be frequency dependent. Iontophoretic application of acetylcholine to the synaptic end of the cell decreases its length. The microarchitecture of the organ of Corti permits length changes of outer hair cells in a manner that could significantly influence the mechanics of the cochlear partition and thereby contribute to the exquisite sensitivity of mammalian hearing.

Synthesis and storage of [3H]acetylcholine in isolated pieces of chick retina increased in two stages during embryogenesis. The first increase coincided with a 100-fold rise in the activity of choline acetyltransferase (acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6), but during the second increase the activity of this enzyme remained essentially constant. The second increase instead was linked to an approximately 6-fold increase in the Vmax for high-affinity uptake of choline.

Intracellular recordings from drone photoreceptors were made by means of glass microelectrodes in superfused retinae. Exposure of a small portion of a cell to white light decreased the amplitude of responses to a small stimulus subsequently applied at different sites of the photoreceptor cell, i.e. light adaptation occurred throughout the cell. After 7 min of darkness, the responses had completely recovered. When a violet light (404 nm) was used to adapt a small portion of the cell, the responses at the site of exposure to the adapting stimulus remained depressed for at least 30 min. Illumination at the site of the violet adapting stimulus with green light (585 nm) caused an immediate recovery of the amplitude of the response. These results can be explained by the existence of two processes responsible for light adaptation: one is localized and persistent and appears to be due to changes in concentration of rhodopsin. The other affects the whole cell, is spontaneously reversible and depends upon the ability of the light to produce a receptor potential but not on any lasting change in rhodopsin concentration.

Solitary rod inner segments were obtained by enzymic dissociation of the tiger salamander retina. Ih, an inward current activated by membrane hyperpolarization, was studied using the single-pipette voltage-clamp technique with patch pipettes. In order to investigate Ih in isolation from voltage-dependent potassium and calcium currents, it was necessary to superfuse with a solution containing TEA and cobalt. When the solution in the patch pipette contained 45 mM-KCl and 50 mM-NaCl, the characteristics of Ih were indistinguishable from those previously described with fine-tip micro-electrodes: the reversal potential was near-30 mV and Ih was blocked by extracellular caesium and enhanced by an increase in the extracellular potassium concentration. The increase in Ih observed when the extracellular potassium concentration is raised is due to an increase in conductance and in driving force. Replacement of sodium in the patch pipette with choline caused a 15 mV displacement of the reversal potential for Ih in the depolarized direction. When using sodium-free patch pipettes, replacement of extracellular sodium displaced the reversal potential for Ih to -74 mV, a value in the range of the potassium equilibrium potential in solitary inner segments. Intracellular or intra- and extracellular sodium substitution affected neither the activation range of Ih nor the maximum conductance. From points 3-6 it can be concluded that Ih is carried mainly, if not exclusively, by sodium and potassium and that the channel responsible for Ih is insensitive to modifications of the intra- or extracellular sodium concentration. The results of long-term hyperpolarization, of partial block with caesium and of total sodium substitution are consistent with sodium and potassium permeating the same type of channel.

Muscle satellite cells play an important role in the postnatal growth of skeletal muscle and in the regeneration of damaged muscle during adult life. Little is known about the physiological properties of satellite cells in their dormant state as they lie adjacent to the intact muscle fibers, underneath the basement membrane. Our recent experiments, using patch clamp techniques, indicate that no tight electrical coupling is present between satellite cells and the muscle fiber dissociated from rat flexor digitorum brevis. Satellite cells possess sodium channels with low sensitivity to tetrodotoxin and at a much lower density than muscle. In addition, satellite cells are insensitive to acetylcholine (ACh) for at least 24 hr after having been removed from the animal, even when detached from their muscle fiber. However, we could measure ACh-evoked currents from satellite cells 48-72 hr in culture, indicating that ACh sensitivity develops with time.

Single, isolated rod photoreceptors were obtained by enzymatic dissociation of the tiger salamander (Ambystoma tigrinum) retina. These solitary cells retained the morphological features of rods of the intact retina and could be maintained in culture for several days. When impaled with micropipettes for electrophysiological recording, dark-adapted solitary rods had during darkness a resting potential of approximately -45 mV and a steady-state slope resistance of 500 Momega at rest. The current-voltage relationship showed both inward- and outward-going rectification. The responses to light of solitary rods were similar to those recorded from rods in the intact retina stimulated with large-diameter spots of light. The reversal potential of the light response of solitary rods was near 0 mV when measured in either the inner or outer segment.

The potassium current of neurons in explants cultured from quail mesencephalic neural crest were studied in voltage clamp, using the whole cell recording technique. Two voltage-dependent potassium currents were identified; they differed in their sensitivity to blocking agents and to sustained depolarizing voltages. The potassium current component most sensitive to 4-aminopyridine had fast activation kinetics and inactivated quickly at sustained depolarized voltages. By analogy with a current described in other preparations, this current was called IA. The current component most sensitive to tetraethylammonium had slower activation kinetics and inactivated more slowly than IA at sustained depolarized voltages. This current was called IK. The properties of IA and IK were examined in neurons cultured in a defined medium and in neurons co-cultured with striated muscle. The rate of inactivation of IA appeared to be increased when neural crest neurons were cultured in the presence of striated muscle. The change in the properties of IA could be due to a direct effect of the co-culture with muscle on the membrane current; another possibility could be that co-culture favors the survival of a neuronal population that does not survive well when cultured in a defined medium.

A chloride current activated by an increase in intracellular calcium concentration is not present in all neurons of the trigeminal ganglion. It is not known whether the trigeminal neurons expressing calcium-activated chloride current belong to a defined class of neurons or whether they could belong to any class of sensory neurons. An answer to this question would be of importance because the physiological role of calcium-activated chloride current in neurons has not yet been completely established, nonetheless it is clear that this current, when activated, would act to modulate neuronal excitability. The goal of this study was to determine whether there was a difference in the expression of calcium-activated chloride current between neurons with and without substance P. The rationale was that the use of this morphological marker, which is present in a substantial fraction of embryonic trigeminal neurons, may give a first estimate of a possible inhomogeneity in the expression of calcium-activated chloride current among different classes of sensory neurons. The study was done on freshly dissociated neurons in order to minimize the influence of the culture conditions on the expression of the current or of substance P. By recording from large samples of neurons in cultures either enriched or depleted in substance P-containing neurons, we found that neurons with substance P expressed calcium-activated chloride current three times less frequently than neurons without substance P. This observation was confirmed by performing the immunocytochemical labelling for substance P immediately after the electrophysiological assessment of the presence or absence of calcium-activated chloride current. This result indicates that calcium-activated chloride current may not be randomly distributed in neurons of a sensory ganglion. It raises the possibility that neurons belonging to certain sensory modalities may need calcium-activated chloride current for their physiological functioning.

Embryonic human spinal cord cells have been grown in dissociated monolayer cultures for 1 to 7 weeks. Using cell type specific markers, it was possible to show that the cultures contain neurons, astrocytes and fibroblasts. Electrical membrane properties were studied with patch electrodes using the whole cell recording technique. Neurons had short duration action potentials that could be blocked by tetrodotoxin. The membrane currents in these neurons were studied in voltage clamp experiments. Three types of voltage-dependent currents were observed: a sodium current; a potassium current made up of two components, IA and IK; and a calcium current. Both cholinergic and GABAergic neurons are present in the cultures. There is more choline acetyltransferase activity in cultures prepared from the anterior as compared to the posterior part of the spinal cord, suggesting that the cultures contain motoneurons. This tissue culture preparation was developed for the study of amyotrophic lateral sclerosis; we have been unable to detect the presence of any toxic agent from the serum of these patients on the cultured cells. Experiments are in progress to purify the motoneurons using Percoll gradients.

The expression of a calcium-activated chloride current (ICl(Ca)) was studied during the development of the sensory neurons of quail trigeminal ganglia. This current is expressed in 20% of the neurons by the 5th day of embryonic development; it can be found in nearly all neurons by the 7th day and subsequently disappears in half of them. Similar results were obtained with dorsal root ganglion neurons. The disappearance of ICl(Ca) in part of the sensory neurons during development is not due to a selective death of the neurons possessing this current and our results suggest that it is mediated by an interaction of the sensory neurons with their target tissue.

A device which can generate rectangular currents in the picoampere range is described. The current generator is a photodiode connected to the head stage of a single-channel recording amplifier. The photodiode is activated by a light-emitting diode controlled by a computer or any other current source. The device can transmit signals corresponding to simulated single-channel behaviour. Since the kinetic parameters of the simulation are known, the user can test the data acquisition and analysis system under conditions similar to those prevailing during recording from a biological membrane. This current generator can also be used for the tuning of patch-clamp amplifiers; rectangular currents generated by the photodiode allow the frequency response of the amplifier to be properly adjusted.

Brain nicotinic acetylcholine receptors (nAChRs) are made up of protein subunits that differ from those constituting muscle nAChRs. To characterize the physiological properties of one class of avian brain nicotinic receptor, we injected the nuclei of Xenopus oocytes with full-length cDNAs for the ligand binding (alpha 4) and structural (n alpha) subunits. Injected oocytes had large ACh-induced currents in the microampere range that were insensitive to alpha-bungarotoxin, as expected for neuronal nAChRs. We found that these brain nAChRs incorporate at least two alpha 4 subunits and that their functional properties differ from muscle nAChRs in at least two respects: the elementary conductance is considerably smaller (20 pS), and channels in outside out patches stop functioning within a few minutes.