Fabio Ruzzier

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-activated potassium channels in chondrocytes

The presence of calcium-activated potassium channels in chondrocytes of growing cartilage was tested. Results obtained with fura-2 on cultured resting chondrocytes indicate that the cells respond to an elevation of extracellular calcium concentration ([Ca2+]o) from 0.1 to 2 mM increasing the intracellular concentration of the ion ([Ca2+]i) from 117 to 187 nM. This increment may be blocked by 3 microM La3+. Patch clamp experiments in cell-attached configuration showed that, when [Ca2+]i rises, the open probability (Po) of the K+ channels increases. Increments in both Po and unitary currents of the K+ channels can be obtained after applying 2.5 microM A23187 with 2 mM [Ca2+]o. Hence, the results demonstrate that, in chondrocytes, a class of Ca(2+)-activated K+ channels is present and their activity is related to an increase of [Ca2+]i.

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.

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.

Properties of glutamate receptors of Alzheimer's disease brain transplanted to frog oocytes

It is known that Alzheimers disease (AD) is a synaptic disease that involves various neurotransmitter systems, particularly those where synaptic transmission is mediated by acetylcholine or glutamate (Glu). Nevertheless, very little is known about the properties of neurotransmitter receptors of the AD human brain. We have shown previously that cell membranes, carrying neurotransmitter receptors from the human postmortem brain, can be transplanted to frog oocytes, and their receptors will still be functional. Taking advantage of this fact, we have now studied the properties of Glu receptors (GluRs) from the cerebral cortices of AD and non-AD brains and found that oocytes injected with AD membranes acquired GluRs that have essentially the same functional properties as those of oocytes injected with membranes from non-AD brains. However, the amplitudes of the currents elicited by Glu were always smaller in the oocytes injected with membranes from AD brains. Western blot analyses of the same membrane preparations used for the electrophysiological studies showed that AD membranes contained significantly fewer GluR2/3 subunit proteins. Furthermore, the corresponding mRNAs were also diminished in the AD brain. Therefore, the smaller amplitude of membrane currents elicited by Glu in oocytes injected with membranes from an AD brain is a consequence of a reduced number of GluRs in cell membranes transplanted from the AD brain. Thus, using the comparatively simple method of microtransplantation of receptors, it is now possible to determine the properties of neurotransmitter receptors of normal and diseased human brains. That knowledge may help to decipher the etiology of the diseases and also to develop new treatments.

A potassium channel in cultured chondrocytes

SummaryChondrocytes, obtained from preosseous cartilage, were studied by patch clamp technique in cell-attached recording configuration, and single potassium channels were characterized at different stages of culture. After 3 days, outward currents were present, with an open probability increasing with depolarization, and the K+ channels showing a mean slope conductance of 82 pS in asymmetric and 168 pS in symmetric potassium solution. Tetraethylammonium (TEA) and quinidine blocked the channels. Cells at confluence showed similar channel activity, with conductances of 121 and 252 pS, respectively. We suggest that culture time and/or conditions may modify K+ channels or induce the expression of a new type of channels.

Effect of repetitive stimulation on the frog neuromuscular transmission

Presynaptic and postsynaptic effects on the neuromuscular transmission were studied during 20 min of indirect stimulation at 10/s. During the ‘facilitation’ period, there was an increase in the quantal content, in the frequency of miniature endplate potentials and in their amplitude. All these parameters were decreased during the ‘depression’ period. Besides, the end-plate current (e.p.c.), recorded during this high rate of stimulation, increasingly lengthened. The falling phase of the e.p.c. was exponential during facilitation, while marked deviations from the exponential time course were observed during depression. The experiments showed that a possible change in the kinetics between the receptors and the mediator was not responsible for the lengthened time course of the e.p.c. Therefore, it is assumed that either the delayed diffusion of the transmitter from the synaptic cleft or an altered mechanism of the release of acetylcholine may be involved. The latter possibility is supported by a progressive prolongation of the synaptic delay, which was observed during a prolonged repetitive stimulation.

Corticosterone modifies the murine muscle acetylcholine receptor channel kinetics

THE biophysical properties of the nicotinic acetylcholine receptor (AChR) are known to be modulated by some steroid hormones; their precise mechanism of action is, however, unclear. A possible direct effect of the glucocorticoid corticosterone (COR) on AChRs of mouse C2C12 myotubes was studied in the cell-attached patchclamp configuration. When added to pipette solution containing acetylcholine, COR had no effect on the single channel conductance, but it reduced the longest time constant of both open time and burst duration histograms by 55% and 65%, respectively. COR also increased nearly by 10-fold the middle time constant of the closed time histogram. COR coupled to a hydrophilic molecule such as bovine serum albumin, however, affected only the closed time distribution. These results suggest the existence of specific recognition sites for COR on the surface of the cell membrane and/or the AChR protein.

A large-conductance voltage-dependent potassium channel in cultured pig articular chondrocytes

Abstract The patch-clamp techniques were used to study voltage-dependent potassium channels in cultured pig articular chondrocytes. A predominant single-channel conductance of 125 pS was found. These channels were reversibly blocked by tetraethylammonium. In cell-attached patches, transient increases in the channel activity were observed, and defined as a switching between low and high activity modes (LAM and HAM). Open-time distributions could be described with two kinetics components (in LAM and HAM) having similar time constants (fast τ1 and slow τ2). In HAM, the area of the slow component was larger. The mean burst length was significantly longer in HAM than in LAM. In both modes, the burst-length distributions were fitted with a sum of three exponentials. In LAM and HAM, the time constants τ1 and τ2 were indistinguishable from those of the open-time distributions. The slowest time constant, τ3, was strongly voltage dependent, and was significantly longer in HAM than in LAM. In both LAM and HAM, the ensemble currents were characterised by a rapid rising phase followed by fast and profound inactivation. The activation kinetics were similar, but the inactivation was faster in HAM. In the outside-out configuration no evidence for mode switching was found. The kinetics of the rising phase of the ensemble currents were also similar to those observed using the cell-attached configuration, but the channels did not inactivate. In the whole-cell configuration, the mode switching was not present. The inactivation time constant showed a large scattering, and was much slower than that measured in the cell-attached patch mode. These currents were blocked by tetraethylammonium and 4-aminopyridine. Our results indicate that intracellular factors are involved in controlling the mode switching and the kinetics of the inactivation of potassium channels in pig articular chondrocytes.