A Pharmacological Investigation of the TMEM16A Currents in Murine Skeletal Myogenic Precursor Cells.

TMEM16A is a Ca2+-activated Cl- channel expressed in various species and tissues. In mammalian skeletal muscle precursors, the activity of these channels is still poorly investigated. Here, we characterized TMEM16A channels and investigated if the pharmacological activation of Piezo1 channels could modulate the TMEM16A currents in mouse myogenic precursors. Whole-cell patch-clamp recordings combined with the pharmacological agents Ani9, T16inh-A01 and Yoda1 were used to characterize TMEM16A-mediated currents and the possible modulatory effect of Piezo1 activity on TMEM16A channels. Western blot analysis was also carried out to confirm the expression of TMEM16A and Piezo1 channel proteins. We found that TMEM16A channels were functionally expressed in fusion-competent mouse myogenic precursors. The pharmacological blockage of TMEM16A inhibited myocyte fusion into myotubes. Moreover, the specific Piezo1 agonist Yoda1 positively regulated TMEM16A currents. The findings demonstrate, for the first time, a sarcolemmal TMEM16A channel activity and its involvement at the early stage of mammalian skeletal muscle differentiation. In addition, the results suggest a possible role of mechanosensitive Piezo1 channels in the modulation of TMEM16A currents.

Neuronal Agrin Promotes Proliferation of Primary Human Myoblasts in an Age-Dependent Manner.

Neuronal agrin, a heparan sulphate proteoglycan secreted by the α-motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts.

Nerve-dependent distribution of subsynaptic type 1 inositol 1,4,5-trisphosphate receptor at the neuromuscular junction.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments of the neuromuscular junction (NMJ), surrounding the subsynaptic nuclei and close to nicotinic acetylcholine receptors (nAChRs) of the motor endplate. At the endplate level, it has been proposed that nerve-dependent electrical activity might trigger IP3-associated, local Ca2+ signals not only involved in excitation-transcription (ET) coupling but also crucial to the development and stabilization of the NMJ itself. The present study was undertaken to examine whether denervation affects the subsynaptic IP3R distribution in skeletal muscles and which are the underlying mechanisms. Fluorescence microscopy, carried out on in vivo denervated muscles (following sciatectomy) and in vitro denervated skeletal muscle fibers from flexor digitorum brevis (FDB), indicates that denervation causes a reduction in the subsynaptic IP3R1-stained region, and such a decrease appears to be determined by the lack of muscle electrical activity, as judged by partial reversal upon field electrical stimulation of in vitro denervated skeletal muscle fibers.

A preliminary study on the role of Piezo1 channels in myokine release from cultured mouse myotubes.

It has long been known that regular physical exercise induces short and long term benefits reducing the risk of cardiovascular disease, diabetes, osteoporosis, cancer and improves sleep quality, cognitive level, mobility, autonomy in enderly. More recent is the evidence on the endocrine role of the contracting skeletal muscle. Exercise triggers the release of miokines, which act in autocrine, paracrine and endocrine ways controlling the activity of muscles but also of other tissues and organs such as adipose tissue, liver, pancreas, bones, and brain. The mechanism of release is still unclear. Neuromuscular electrical stimulation reproduces the beneficial effects of physical activity producing physiological metabolic, cardiovascular, aerobic responses consistent with those induced by exercise. In vitro, Electrical Pulse Stimulations (EPS) of muscle cells elicit cell contraction and mimic miokine release in the external medium. Here we show that, in cultured mouse myotubes, EPS induce contractile activity and the release of the myokine IL-6. Gadolinium highly reduces EPS-induced IL-6 release, suggesting the involvement of mechanical activated ion channels. The chemical activation of mechanosensitive Piezo1 channels with the specific agonist Yoda1 stimulates IL-6 release similarly to EPS, suggesting the involvement of Piezo1 channels in the control of the myokine release. The expression of Piezo1 protein in myotubes was confirmed by the Western blot analysis. To the best of our knowledge, this is the first evidence of a Piezo1-mediated effect in myokine release and suggests a potential translational use of specific Piezo1 agonists for innovative therapeutic treatments reproducing/enhancing the benefits of exercise mediated by myokines.

The State of the Art of Piezo1 Channels in Skeletal Muscle Regeneration.

Piezo1 channels are highly mechanically-activated cation channels that can sense and transduce the mechanical stimuli into physiological signals in different tissues including skeletal muscle. In this focused review, we summarize the emerging evidence of Piezo1 channel-mediated effects in the physiology of skeletal muscle, with a particular focus on the role of Piezo1 in controlling myogenic precursor activity and skeletal muscle regeneration and vascularization. The disclosed effects reported by pharmacological activation of Piezo1 channels with the selective agonist Yoda1 indicate a potential impact of Piezo1 channel activity in skeletal muscle regeneration, which is disrupted in various muscular pathological states. All findings reported so far agree with the idea that Piezo1 channels represent a novel, powerful molecular target to develop new therapeutic strategies for preventing or ameliorating skeletal muscle disorders characterized by an impairment of tissue regenerative potential.

Preliminary Observations on Skeletal Muscle Adaptation and Plasticity in Homer 2-/- Mice.

Homer represents a diversified family of scaffold and transduction proteins made up of several isoforms. Here, we present preliminary observations on skeletal muscle adaptation and plasticity in a transgenic model of Homer 2-/- mouse using a multifaceted approach entailing morphometry, quantitative RT-PCR (Reverse Transcription PCR), confocal immunofluorescence, and electrophysiology. Morphometry shows that Soleus muscle (SOL), at variance with Extensor digitorum longus muscle (EDL) and Flexor digitorum brevis muscle (FDB), displays sizable reduction of fibre cross-sectional area compared to the WT counterparts. In SOL of Homer 2-/- mice, quantitative RT-PCR indicated the upregulation of Atrogin-1 and Muscle ring finger protein 1 (MuRF1) genes, and confocal immunofluorescence showed the decrease of neuromuscular junction (NMJ) Homer content. Electrophysiological measurements of isolated FDB fibres from Homer 2-/- mice detected the exclusive presence of the adult ε-nAChR isoform excluding denervation. As for NMJ morphology, data were not conclusive, and further work is needed to ascertain whether the null Homer 2 phenotype induces any endplate remodelling. Within the context of adaptation and plasticity, the present data show that Homer 2 is a co-regulator of the normotrophic status in a muscle specific fashion.

"Time window" effect of Yoda1-evoked Piezo1 channel activity during mouse skeletal muscle differentiation.

AIM: Mechanosensitive Piezo1 ion channels emerged recently as important contributors to various vital functions including modulation of the blood supply to skeletal muscles. The specific Piezo1 channel agonist Yoda1 was shown to regulate the tone of blood vessels similarly to physical exercise. However, the direct role of Piezo1 channels in muscle function has been little studied so far. We therefore investigated the action of Yoda1 on the functional state of skeletal muscle precursors (satellite cells and myotubes) and on adult muscle fibres. METHODS: Immunostaining, electrophysiological intracellular recordings and Ca2+ imaging experiments were performed to localize and assess the effect of the chemical activation of Piezo1 channels with Yoda1, on myogenic precursors, adult myofibres and at the adult neuromuscular junction. RESULTS: Piezo1 channels were detected by immunostaining in satellite cells (SCs) and myotubes as well as in adult myofibres. In the skeletal muscle precursors, Yoda1 treatment stimulated the differentiation and cell fusion rather than the proliferation of SCs. Moreover, in myotubes, Yoda1 induced significant [Ca2+ ]i transients, without detectable [Ca2+ ]i response in adult myofibres. Furthermore, although expression of Piezo1 channels was detected around the muscle endplate region, Yoda1 application did not alter either the nerve-evoked or spontaneous synaptic activity or muscle contractions in adult myofibres. CONCLUSION: Our data indicate that the chemical activation of Piezo1 channels specifically enhances the differentiation of skeletal muscle precursors, suggesting a possible new strategy to promote muscle regeneration.

Interplay Between Cholinergic and Adenosinergic Systems in Skeletal Muscle.

Since the pioneering works of Ricardo Miledi, the neuromuscular junction represents the best example of a synapse where ACh is the neurotransmitter acting on nicotinic ACh receptors. ATP, co-released with ACh, is promptly degraded to Ado, which acts as a modulator of the cholinergic synaptic activity. Consequently, both ACh and adenosine play a crucial role in controlling the nerve-muscle communication. Apart from their role in the context of synaptic transmission, ACh and adenosine are autocrinally released by skeletal muscle cells, suggesting also a non nerve-driven function of these molecules. Indeed, the existence of cholinergic and adenosinergic systems has been widely described in many other non neuronal cell types. In this review, we will describe the two systems and their interplay in non-innervated differentiating skeletal muscle cells, and in innervated adult skeletal muscle fibers. We believe that the better comprehension of the interactions between the activity of nAChRs and adenosine could help the knowledge of skeletal muscle physiology. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

A "noisy" electrical stimulation protocol favors muscle regeneration in vitro through release of endogenous ATP.

An in vitro system of electrical stimulation was used to explore whether an innovative "noisy" stimulation protocol derived from human electromyographic recordings (EMGstim) could promote muscle regeneration. EMGstim was delivered to cultured mouse myofibers isolated from Flexor Digitorum Brevis, preserving their satellite cells. In response to EMGstim, immunostaining for the myogenic regulatory factor myogenin, revealed an increased percentage of elongated myogenin-positive cells surrounding the myofibers. Conditioned medium collected from EMGstim-treated cell cultures, promoted satellite cells differentiation in unstimulated myofiber cell cultures, suggesting that extracellular soluble factors could mediate the process. Interestingly, the myogenic effect of EMGstim was mimicked by exogenously applied ATP (0.1 µM), reduced by the ATP diphosphohydrolase apyrase and prevented by blocking endogenous ATP release with carbenoxolone. In conclusion, our results show that "noisy" electrical stimulations favor muscle progenitor cell differentiation most likely via the release of endogenous ATP from contracting myofibres. Our data also suggest that "noisy" stimulation protocols could be potentially more efficient than regular stimulations to promote in vivo muscle regeneration after traumatic injury or in neuropathological diseases.

Epidermal Growth Factor - based adhesion substrates elicit myoblast scattering, proliferation, differentiation and promote satellite cell myogenic activation.

The biochemical properties of muscle extracellular matrix are essential for stem cell adhesion, motility, proliferation and myogenic development. Recombinant elastin-like polypeptides are synthetic polypeptides that, besides maintaining some properties of the native protein, can be tailored by fusing bioactive sequences to their C-terminal. Our laboratory synthesized several Human Elastin-Like Polypeptides (HELP) derived from the sequence of human tropoelastin. Here, we developed a novel HELP family member by fusing the elastin-like backbone to the sequence of human Epidermal Growth Factor. We employed this synthetic protein, named HEGF, either alone or in combination with other proteins of the HELP family carrying RGD-integrin binding sites, as adhesion substrate for C2C12 myoblasts and satellite cells primary cultures. Adhesion of myoblasts to HEGF-based substrates induced scattering, decreased adhesion and cytoskeleton assembly; the concomitant presence of the RGD motifs potentiated all these effects. Recombinant substrates induced myoblasts proliferation, differentiation and the development of multinucleated myotubes, thus favoring myoblasts expansion and preserving their myogenic potential. The effects induced by adhesion substrates were inhibited by AG82 (Tyrphostin 25) and herbimycin A, indicating their dependence on the activation of both the EGF receptor and the tyrosine kinase c-src. Finally, HEGF increased the number of muscle stem cells (satellite cells) derived from isolated muscle fibers in culture, thus highlighting its potential as a novel substrate for skeletal muscle regeneration strategies.

Adenosine Promotes Endplate nAChR Channel Activity in Adult Mouse Skeletal Muscle Fibers via Low Affinity P1 Receptors.

Adenosine is a powerful modulator of skeletal neuromuscular transmission, operating via inhibitory or facilitatory purinergic-type P1 receptors. To date, studies have been focused mainly on the effect of adenosine on presynaptic P1 receptors controlling transmitter release. In this study, using two-microelectrode voltage-clamp and single-channel patch-clamp recording techniques, we have explored potential postsynaptic targets of adenosine and their modulatory effect on nicotinic acetylcholine receptor (nAChR)-mediated synaptic responses in adult mouse skeletal muscle fibers in vitro. In the whole-mount neuromuscular junction (NMJ) preparation, adenosine (100 µM) significantly reduced the frequency of the miniature endplate currents (MEPCs) and slowed their rising and decay time. Consistent with a postsynaptic site of action, adenosine and the potent P1 receptor agonist NECA significantly increased the open probability, the frequency and the open time of single nAChR channels, recorded at the endplate region. Using specific ligands for the P1 receptor subtypes, we found that the low-affinity P1 receptor subtype A2B was responsible for mediating the effects of adenosine on the nAChR channel openings. Our data suggest that at the adult mammalian NMJ, adenosine acts not only presynaptically to modulate acetylcholine transmitter release, but also at the postsynaptic level, to enhance the activity of nAChRs. Our findings open a new scenario in understanding of purinergic regulation of nAChR activity at the mammalian endplate region.

In Vitro Innervation as an Experimental Model to Study the Expression and Functions of Acetylcholinesterase and Agrin in Human Skeletal Muscle.

Acetylcholinesterase (AChE) and agrin, a heparan-sulfate proteoglycan, reside in the basal lamina of the neuromuscular junction (NMJ) and play key roles in cholinergic transmission and synaptogenesis. Unlike most NMJ components, AChE and agrin are expressed in skeletal muscle and α-motor neurons. AChE and agrin are also expressed in various other types of cells, where they have important alternative functions that are not related to their classical roles in NMJ. In this review, we first focus on co-cultures of embryonic rat spinal cord explants with human skeletal muscle cells as an experimental model to study functional innervation in vitro. We describe how this heterologous rat-human model, which enables experimentation on highly developed contracting human myotubes, offers unique opportunities for AChE and agrin research. We then highlight innovative approaches that were used to address salient questions regarding expression and alternative functions of AChE and agrin in developing human skeletal muscle. Results obtained in co-cultures are compared with those obtained in other models in the context of general advances in the field of AChE and agrin neurobiology.

Developmental and maintenance defects in Rett syndrome neurons identified by a new mouse staging system in vitro.

Rett Syndrome (RTT) is a neurodevelopmental disorder associated with intellectual disability, mainly caused by loss-of-function mutations in the MECP2 gene. RTT brains display decreased neuronal size and dendritic arborization possibly caused by either a developmental failure or a deficit in the maintenance of dendritic arbor structure. To distinguish between these two hypotheses, the development of Mecp2-knockout mouse hippocampal neurons was analyzed in vitro. Since a staging system for the in vitro development of mouse neurons was lacking, mouse and rat hippocampal neurons development was compared between 1-15 days in vitro (DIV) leading to a 6-stage model for both species. Mecp2-knockout hippocampal neurons displayed reduced growth of dendritic branches from stage 4 (DIV4) onwards. At stages 5-6 (DIV9-15), synapse number was lowered in Mecp2-knockout neurons, suggesting increased synapse elimination. These results point to both a developmental and a maintenance setback affecting the final shape and function of neurons in RTT.

Toxicity of palytoxin after repeated oral exposure in mice and in vitro effects on cardiomyocytes.

Palytoxin (PLTX) is a highly toxic hydrophilic polyether detected in several edible marine organisms from intra-tropical areas, where seafood poisoning were reported. Symptoms usually start with gastro-intestinal malaise, often accompanied by myalgia, muscular cramps, dyspnea and, sometimes, arrhythmias. Monitoring programs in the Mediterranean Sea have detected PLTX-like molecules in edible mollusks and echinoderms. Despite the potential exposure of the human population and its high toxic potential, the toxicological profile of the molecule is still an issue. Thus, the effects of repeated oral administration of PLTX in mice were investigated. Seven days of PLTX administration caused lethality and toxic effects at doses ≥ 30 µg/kg/day. A NOAEL was estimated equal to 3 µg/kg/day, indicating a quite steep dose-response curve. This value, due to the limited number of animal tested, is provisional, although represents a sound basis for further testing. Macroscopic alterations at gastrointestinal level (gastric ulcers and intestinal fluid accumulation) were observed in mice dead during the treatment period. Histological analysis highlighted severe inflammation, locally associated with necrosis, at pulmonary level, as well as hyper-eosinophilia and fiber separation in myocardium. A cardiac damage was supported by the in vitro effect of the toxin on cardiomyocytes, indicating a severe and irreversible impairment of their electrical properties: electrophysiological recordings detected a progressive cell depolarization, arrest of action potentials and beating.

Acetylcholinesterase and agrin: different functions, similar expression patterns, multiple roles.

Acetylcholinesterase (AChE) and agrin play unique functional roles in the neuromuscular junction (NMJ). AChE is a cholinergic and agrin a synaptogenetic component. In spite of their different functions, they share several common features: their targeting is determined by alternative splicing; unlike most other NMJ components they are expressed in both, muscle and motor neuron and both reside on the synaptic basal lamina of the NMJ. Also, both were reported to play various nonjunctional roles. However, while the origin of basal lamina bound agrin is undoubtedly neural, the neural origin of AChE, which is anchored to the basal lamina with collagenic tail ColQ, is elusive. Hypothesizing that motor neuron proteins targeted to the NMJ basal lamina share common temporal pattern of expression, which is coordinated with the formation of basal lamina, we compared expression of agrin isoforms with the expression of AChE-T and ColQ in the developing rat spinal cord at the stages before and after the formation of NMJ basal lamina. Cellular origin of AChE-T and agrin was determined by in situ hybridization and their quantitative levels by RT PCR. We found parallel increase in expression of the synaptogenetic (agrin 8) isoform of agrin and ColQ after the formation of basal lamina supporting the view that ColQ bound AChE and agrin 8 isoform are destined to the basal lamina. Catalytic AChE-T subunit and agrin isoforms 19 and 0 followed different expression patterns. In accordance with the reports of other authors, our investigations also revealed various alternative functions for AChE and agrin. We have already demonstrated participation of AChE in myoblast apoptosis; here we present the evidence that agrin promotes the maturation of heavy myosin chains and the excitation-contraction coupling. These results show that common features of AChE and agrin extend to their capacity to play multiple roles in muscle development.

Reactive oxygen species contribute to the promotion of the ATP-mediated proliferation of mouse skeletal myoblasts.

Reactive oxygen species (ROS) and extracellular adenosine 5'-triphosphate (ATP) participate in autocrine and paracrine regulation in skeletal muscle. However, the link between these two signaling systems is not well established. Here, we studied cell proliferation as a possible consequence of the trophic effect of ATP in cultured skeletal mouse myoblasts and we tested the possibility that low concentrations of ROS represent the intermediate signaling molecule mediating this effect. Exposure to 10 µM ATP increased proliferation of mouse myoblasts by ~20%. ATP also induced intracellular Ca(2+) oscillations, which were independent of extracellular Ca(2+). Both effects of ATP were prevented by suramin, a broad-spectrum purinergic P2 receptor antagonist. In contrast, the adenosine receptor blocker CGS-15943 did not modify the ATP-mediated effects. Consistent with this, adenosine per se did not change myoblast growth, indicating the direct action of ATP via P2 receptor activation. The proliferative effect of ATP was prevented after depletion of hydrogen peroxide (H(2)O(2)) by the peroxidase enzyme catalase. Low-micromolar concentrations of exogenous H(2)O(2) mimicked the stimulatory effect of ATP on myoblast growth. DCF imaging revealed ATP-induced catalase and DPI-sensitive ROS production in myoblasts. In conclusion, our results indicate that extracellular ATP controls mouse myoblast proliferation via induction of ROS generation.

Physical exercise and antidepressants enhance BDNF targeting in hippocampal CA3 dendrites: further evidence of a spatial code for BDNF splice variants.

Brain-derived neurotrophic factor (BDNF) is encoded by multiple BDNF transcripts, whose function is unclear. We recently showed that a subset of BDNF transcripts can traffic into distal dendrites in response to electrical activity, while others are segregated into the somatoproximal domains. Physical exercise and antidepressant treatments exert their beneficial effects through upregulation of BDNF, which is required to support survival and differentiation of newborn dentate gyrus (DG) neurons. While these DG processes are required for the antidepressant effect, a role for CA1 in antidepressant action has been excluded, and the effect on CA3 neurons remains unclear. Here, we show for the first time that physical exercise and antidepressants induce local increase of BDNF in CA3. Voluntary physical exercise for 28 consecutive days, or 2-week treatment with 10 mg/kg per day fluoxetine or reboxetine, produced a global increase of BDNF mRNA and protein in the neuronal somata of the whole hippocampus and a specific increase of BDNF in dendrites of CA3 neurons. This increase was accounted for by BDNF exon 6 variant. In cultured hippocampal neurons, application of serotonin or norepinephrine (10-50 µM) induced increase in synaptic transmission and targeting of BDNF mRNA in dendrites. The increased expression of BDNF in CA3 dendrites following antidepressants or exercise further supports the neurotrophin hypothesis of antidepressants action and confirms that the differential subcellular localization of BDNF mRNA splice variants provides a spatial code for a selective expression of BDNF in specific subcellular districts. This selective expression may be exploited to design more specific antidepressants.

Nestin- and doublecortin-positive cells reside in adult spinal cord meninges and participate in injury-induced parenchymal reaction.

Adult spinal cord has little regenerative potential, thus limiting patient recovery following injury. In this study, we describe a new population of cells resident in the adult rat spinal cord meninges that express the neural stem/precursor markers nestin and doublecortin. Furthermore, from dissociated meningeal tissue a neural stem cell population was cultured in vitro and subsequently shown to differentiate into functional neurons or mature oligodendrocytes. Proliferation rate and number of nestin- and doublecortin-positive cells increased in vivo in meninges following spinal cord injury. By using a lentivirus-labeling approach, we show that meningeal cells, including nestin- and doublecortin-positive cells, migrate in the spinal cord parenchyma and contribute to the glial scar formation. Our data emphasize the multiple roles of meninges in the reaction of the parenchyma to trauma and indicate for the first time that spinal cord meninges are potential niches harboring stem/precursor cells that can be activated by injury. Meninges may be considered as a new source of adult stem/precursor cells to be further tested for use in regenerative medicine applied to neurological disorders, including repair from spinal cord injury.

Effects of H2O2 on electrical membrane properties of skeletal myotubes.

Reactive oxygen species (ROS), normally generated in skeletal muscles, could control excitability of muscle fibers through redox modulation of membrane ion channels. However, the mechanisms of ROS action remain largely unknown. To investigate the action of ROS on electrical properties of muscle cells, patch-clamp recordings were performed after application of hydrogen peroxide (H2O2) to skeletal myotubes. H2O2 facilitated sodium spikes after a hyperpolarizing current pulse, by decreasing the latency for spike initiation. Importantly, the antioxidant N-acetylcysteine induced the opposite effect, suggesting the redox control of muscle excitability. The effect of H2O2 was abolished in the presence of catalase. The kinetics of sodium channels were not affected by H2O2. However, the fast inward rectifier K(+) (K(IR)) currents, activated by hyperpolarization, were reduced by H2O2, similar to the action of the potassium channel blockers Ba(2+) and Cs(+). The block of the outward tail current contributing to K(IR) deactivation can explain the shorter latency for spike initiation. We propose that the K(IR) current is an important target for ROS action in myotubes. Our data would thus suggest that ROS are involved in the control of the excitability of myotubes and, possibly, in the oscillatory behavior critical for the plasticity of developing muscle cells.

Neural agrin changes the electrical properties of developing human skeletal muscle cells.

Recent investigations suggest that the effects of neural agrin might not be limited to neuromuscular junction formation and maintenance and that other aspects of muscle development might be promoted by agrin. Here we tested the hypothesis that agrin induces a change in the excitability properties in primary cultures of non-innervated human myotubes. Electrical membrane properties of human myotubes were recorded using the whole-cell patch-clamp technique. Cell incubation with recombinant chick neural agrin (1 nM) led to a more negative membrane resting potential. Addition of strophanthidin, a blocker of the Na(+)/K(+) ATPase, depolarized agrin-treated myotubes stronger than control, indicating, in the presence of agrin, a higher contribution of the Na(+)/K(+) ATPase in establishing the resting membrane potential. Indeed, larger amounts of both the alpha1 and the alpha2 isoforms of the Na(+)/K(+) ATPase protein were expressed in agrin-treated cells. A slight but significant down-regulation of functional apamin-sensitive K(+) channels was observed after agrin treatment. These results indicate that neural agrin might act as a trophic factor promoting the maturation of membrane electrical properties during differentiation, confirming the role of agrin as a general promoter of muscle development.