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.

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. 3-(4,5-Dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) and sulforhodamine B (SRB) tests were carried out to determine its effect on cardiomyocytes viability. Videoimaging techniques showed a time- and concentration-dependent reduction in the beating frequency after 1, 5, and 24 h incubation with YTX (0.1-1 microM). This effect was neither associated to the uncoupling between the membrane electrical activity and Ca(2+) release from intracellular stores nor to the impairment of the mechanisms controlling the Ca(2+) homeostasis. In addition, 1 microM YTX did not modify basal cAMP levels in cardiomyocytes. MTT and SRB assays revealed that incubation of cardiomyocytes with YTX (0.01-1 microM; 24, 48, and 72 h) caused a decrease in cell viability in a concentration- and time-dependent way. This effect was still evident in cardiomyocytes exposed to YTX for 1, 5, and 24 h and cultured up to 72 h in YTX-free medium. Our results demonstrate that, at nanomolar concentrations, a short incubation with YTX causes an inhibition of the beating activity and an irreversible reduction of viability of cardiac cells in vitro.

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. The effect of spinal cord explants was mimicked by treatment with medium conditioned by cocultures or by addition of 1 nM of recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors (RyRs) and dihydropyridine-sensitive L-type Ca(2+) channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both RyRs and L-type Ca(2+) channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.

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.