Paola Lorenzon

Ageing affects the differentiation potential of human myoblasts

The ageing process causes a reduction in the regenerative potential of skeletal muscles eventually leading to diminished muscle strength. In this work we investigated if ageing affects the excitation-contraction coupling mechanism in human myotubes derived from human satellite cells, thereby contributing to the loss in muscle strength in the aged. To test this hypothesis, satellite cells from differently aged donors were differentiated in vitro and the maturation of the excitation-contraction mechanism was followed by the videoimaging technique monitoring the efficiency of such a mechanism in generating intracellular calcium transients. Our experiments showed a delay in the establishment of the excitation-contraction coupling mechanism depending on the age of the donor. Remarkably, the effect was reproducible in human satellite cells from a young donor aged in vitro, suggesting that the delayed functional maturation was strictly dependent on the number of satellite cell divisions and independent from the host environment.

SPONTANEOUS AND REPETITIVE CALCIUM TRANSIENTS IN C2C12 MOUSE MYOTUBES DURING IN VITRO MYOGENESIS

Fluorescence videomicroscopy was used to monitor changes in the cytosolic free Ca2+ concentration ([Ca2+]i in the mouse muscle cell line C2C12 during in vitro myogenesis. Three different patterns of changes in [Ca2+]i were observed: (i) [Ca2+]i oscillations; (ii) faster Ca2+ events confined to subcellular regions (localized [Ca2+]i spikes) and (iii) [Ca2+]i spikes detectable in the entire myotube (global [Ca2+]i spikes). [Ca2+]i oscillations and localized [Ca2+]i spikes were detectable following the appearance of caffeine‐sensitivity in differentiating C2C12 cells. Global [Ca2+]i spikes appeared later in the process of myogenesis in cells exhibiting coupling between voltage‐operated Ca2+ channels and ryanodine receptors. In contrast to [Ca2+]i oscillations and localized [Ca2+]i spikes, the global events immediately stopped when cells were perfused either with a Ca2+‐free solution, or a solution with TTX, TEA and verapamil. To explore further the mechanism of the global [Ca2+]i spikes, membrane currents and fluorescence signals were measured simultaneously. These experiments revealed that global [Ca2+]i spikes were correlated with an inward current. Moreover, while the depletion of the Ca2+ stores blocked [Ca2+]i oscillations and localized [Ca2]i spikes, it only reduced the amplitude of global [Ca2+]i spikes. It is suggested that, during the earlier stages of the myogenesis, spontaneous and repetitive [Ca2+]i changes may be based on cytosolic oscillatory mechanisms. The coupling between voltage‐operated Ca2+ channels and ryanodine receptors seems to be the prerequisite for the appearance of global [Ca2+]i spikes triggered by a membrane oscillatory mechanism, which characterizes the later phases of the myogenic process.

Calcium and Fos Involvement in Brain‐Derived Ca2+‐Binding Protein (S100)‐Dependent Apoptosis in Rat Phaeochromocytoma Cells

Brain‐derived calcium‐binding protein S100 induces apoptosis in a significant fraction of rat phaeochromocytoma (PC12) cells. We used single cell techniques (patch clamp, videomicroscopy and immunocytochemistry) to clarify some of the specific aspects of S100‐induced apoptosis, the modality(ies) of early intracellular Ca2+ concentration increase and the expression of some classes of genes (c‐fos, c‐jun, bax, bcl‐x, p‐15, p‐21) known to be implicated in apoptosis of different cells. The results show that S100: (1) causes an increase of [Ca2+]i due to an increased conductance of L‐type Ca2+ channels; (2) induces a sustained increase of the Fos levels which is evident since the first time point tested (3 h) and remains elevated until to the last time point (72 h). All these data suggest that S100‐derived apoptosis in PC12 cells may be the consequence of a system involving an increase in L‐type Ca2+ channel conductance with consequent [Ca2+]i increase which up‐regulates, directly or indirectly, the expression of Fos.

Autocrine activation of nicotinic acetylcholine receptors contributes to Ca2+ spikes in mouse myotubes during myogenesis

It is widely accepted that nicotinic acetylcholine receptor (nAChR) channel activity controls myoblast fusion into myotubes during myogenesis. In this study we explored the possible role of nAChR channels after cell fusion in a murine cell model. Using videoimaging techniques we showed that embryonic muscle nAChR channel openings contribute to the spontaneous transients of intracellular concentration of Ca2+ ([Ca2+]i) and to twitches characteristic of developing myotubes before innervation. Moreover, we observed a choline acetyltransferase immunoreactivity in the myotubes and we detected an acetylcholine‐like compound in the extracellular solution. Therefore, we suggest that the autocrine activation of nAChR channels gives rise to [Ca2+]i spikes and contractions. Spontaneous openings of the nAChR channels may be an alternative, although less efficient, mechanism. We report also that blocking the nAChRs causes a significant reduction in cell survival, detectable as a decreased number of myotubes in culture. This led us to hypothesize a possible functional role for the autocrine activation of the nAChRs. By triggering mechanical activity, such activation could represent a strategy to ensure the trophism of myotubes in the absence of nerves.

Characterization of specific GTP binding sites in C2C12 mouse skeletal muscle cells

Receptor sites, specific for guanosine 5′-triphosphate (GTP) were characterised in myoblasts and myotubes of C2C12 mouse skeletal muscle cells, using binding experiments and measurements of intracellular Ca2+ concentration ([Ca2+]i). We identified two GTP binding sites in myoblasts membranes: a high affinity site (Kd = 15.4 ± 4.6 μM; Bmax = 1.7 ± 0.5 nmol mg−1 protein); and a low affinity site (Kd = 170 ± 94.5 μM; Bmax = 14.2 ± 3.9 nmol mg−1 protein). In myotube membranes only a low affinity binding site for GTP (Kd = 169 ± 39 μM; Bmax = 12.3 ± 1.4 nmol mg−1 protein) was detected. In myoblasts GTP binding was not displaced by ATP or UTP, even at high concentrations (up to of 1 mM), but it was affected by treatments with suramin or Reactive Blue 2 (RB2), the non-selective purine receptor antagonists. In contrast, in myotubes GTP binding was partially displaced by high concentrations of ATP, but treatments with the non-selective purine receptor antagonists, suramin or RB2, and with UTP had no effect on GTP binding. The addition of GTP to myoblasts, and to myotubes, resulted in elevations of [Ca2+]i. The patterns of Ca2+ response however, were different in the two cell phenotypes. In myoblasts the addition of GTP induced two types of Ca2+ responses: (1) a fast increase in [Ca2+]i, followed by a sustained [Ca2+]i elevation, and (2) a slow raising and steady prolonged increase in [Ca2+]i. In myotubes, however only fast Ca2+ responses were observed following the addition of 500 μM GTP. In the myoblasts and myotubes GTP-stimulated [Ca2+]i increases were abolished by treatments with suramin or RB2 at concentrations which had no effect on the ATP-induced Ca2+ responses. We conclude, that C2C12 cells express two distinct binding sites for GTP before differentiation, but only one after, the low affinity binding site. These results suggest a possible role of the high affinity GTP binding site in early stage of development of skeletal muscle.

Voltage- and ligand-gated ryanodine receptors are functionally separated in developing C2C12 mouse myotubes

1 In order to further understand the role of voltage‐ and ligand‐gated ryanodine receptors in the control of intracellular Ca2+ signalling during myogenesis, changes in cytosolic free calcium concentration ([Ca2+]i) were investigated by fura‐2 videoimaging in C2C12 mouse myotubes developing in vitro. 2 A synchronous [Ca2+]i increase was observed after depolarisation with high [K+], while the Ca2+ response propagated as a wave following caffeine administration. Application of the two stimuli to the same myotube often revealed the existence of cellular zones that were responsive to depolarisation but not to caffeine. 3 Focal application of high [K+] promoted a [Ca2+]i response detectable only in the cellular areas close to the pipette tip, while focal application of caffeine elicited a [Ca2+]i increase which spread as a Ca2+ wave. Buffering of [Ca2+]i by BAPTA did not affect the pattern of the depolarisation‐induced [Ca2+]i transient but abolished the Ca2+ waves elicited by caffeine. 4 When high [K+] and caffeine were applied in sequence, reciprocal inhibition of the [Ca2+]i responses was observed. 5 Our results suggest that the different spatial patterns of [Ca2+]i responses are due to uneven distribution of voltage‐ and ligand‐gated ryanodine receptors within the myotube. These two types of receptor control two functionally distinct Ca2+ pools which are part of a common intracellular compartment. Finally, the two differently operated ryanodine receptor channels appear to be independently activated, so that a mechanism of Ca2+‐induced Ca2+ release is not required to sustain the global response in C2C12 myotubes.

Sanitary problems related to the presence of Ostreopsis spp. in the Mediterranean Sea: a multidisciplinary scientific approach

The increased presence of potentially toxic microalgae in the Mediterranean area is a matter of great concern. Since the end of the last century, microalgae of the genus Ostreopsis have been detected more and more frequently in the Italian coastal waters. The presence of Ostreopsis spp. has been accompanied by the presence of previously undetected marine biotoxins (palytoxins) into the ecosystem with the increased possibility of human exposure. In response to the urgent need for toxicity characterization of palytoxin and its congeners, an integrated study encompassing both in vitro and in vivo methods was performed.

Interleukin-2 lengthens extrajunctional acetylcholine receptor channel open time in mammalian muscle cells

The effect of interleukin-2 (rIL-2) on nicotinic acetylcholine receptors (nAChR) was examined on cultured muscle fibres isolated from the flexor digitorum brevis muscle (FDB) of the rat and on aneural mouse cultured C2 myotubes. Intracellular measurement of the sensitivity to iontophoretically applied ACh demonstrated that the sensitivity of the extrajunctional nAChRs in cultured fibres showed a transient increase after application of rIL-2 (2,000–3,000 units/ml). Cell-attached patch-clamp experiments on the same fibres proved that rIL-2 (2,000 units/ml) induces a significant increase in the mean open time of the extrajunctional nAChR channel. The other channel parameters were not significantly modified. The same applied also to aneural mouse patch-clamped C2 myotubes exposed to rIL-2 (2,000 units/ml). In freshly dissociated fibres no effects on nAChR channels were observed following rIL-2 application. 125I-rIL-2 binding experiments on either 7-day cultured or freshly dissociated adult muscle fibres showed that a specific binding with a Kd of 2.07±0.4 nM develops in cultured fibres but fails to occur immediately after dissociation. It is concluded that rIL-2 modulates the duration of extrajunctional nAChR channels in both myotubes and adult muscle cells, and that this effect is probably due to the activation of a second messenger system.

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.

Mast cell adhesion induces cytoskeletal modifications and programmed cell death in oligodendrocytes

In this paper we show that rat peritoneal mast cells (RPMC) adhere to rat oligodendrocytes (ODC) in culture and switch on a bi-directional signal affecting both adhering cell and its target. Following heterotypic interaction, RPMC release granule content and ODC show morphological changes and enter the apoptotic programme. Altogether, these findings indicate that the interaction of MC with ODC could play a role in the mechanism of CNS damage induced by the inflammatory reaction.