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

TMEM16A is a Ca-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.

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.

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.

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 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.

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 muscle (SOL), at variance with muscle (EDL) and muscle (FDB), displays sizable reduction of fibre cross-sectional area compared to the WT counterparts.

"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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

In vitro effects of yessotoxin on a primary culture of rat cardiomyocytes.

Oral administration of yessotoxin (YTX) has been reported to induce ultrastructural alterations in rodent cardiac muscle. To study its effects on various fundamental aspects of cardiac muscle cells activity, that is, cell beating, Ca(2+) and cyclic adenosine 3',5'-monophosphate (cAMP) levels, as well as cell vitality, a primary culture of rat cardiomyocytes was used. Patch-clamp recordings, Ca(2+) imaging, and cAMP assays were performed on cultured cardiomyocytes to characterize YTX effects on the cell beating frequency.

Neural agrin controls maturation of the excitation-contraction coupling mechanism in human myotubes developing in vitro.

The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: 1) aneurally cultured, 2) cocultured with fetal rat spinal cord explants, 3) aneurally cultured in medium conditioned by cocultures, and 4) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca(2+) imaging indicated that coculturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism.

Intrinsic ionic conductances mediate the spontaneous electrical activity of cultured mouse myotubes.

Mouse skeletal myotubes differentiated in vitro exhibited spontaneous contractions associated with electrical activity. The ionic conductances responsible for the origin and modulation of the spontaneous activity were examined using the whole-cell patch-clamp technique and measuring [Ca(2+)](i) transients with the Ca(2+) indicator, fura 2-AM. Regular spontaneous activity was characterized by single TTX-sensitive action potentials, followed by transient increases in [Ca(2+)](i).

Myocytes from congenital myotonic dystrophy display abnormal Na+ channel activities.

Na(+) currents were measured in myocytes from a fetus with congenital myotonic dystrophy type 1 (DM1) using the patch-clamp whole-cell technique. Steady-state activation and inactivation properties of Na(+) channels were not substantially different between these cells and age-matched control cells. However, a decrease in Na(+) channel density and a faster rate of recovery from inactivation were found in myocytes from congenital DM1 suggesting that changes in functional Na(+) channels may affect cell excitability of muscle cells of patients with this disorder.

gamma-Aminobutyric acidA rho receptor subunits in the developing rat hippocampus.

The RT-PCR approach was used to estimate the expression of gamma-aminobutyric acid (GABA)(A) rho receptor subunits in the hippocampus of neonatal and adult rats. All three rho subunits were detected at postnatal day (P) 2, the rho3 subunit being expressed at an extremely low level. The rho1 and rho2 products appeared to be developmentally regulated; they were found to be more pronounced in adulthood.