Regulation of cytosolic Ca2+ in clonal human muscle cell cultures

doi:10.1016/0006-291X(89)92720-4

Biochemical and Biophysical Research Communications

Volume 165, Issue 3, 30 December 1989, Pages 1130-1137

https://doi.org/10.1016/0006-291X(89)92720-4 Get rights and content

Abstract

Human muscle cells were grown in culture and clonally selected for fusion potential. The concentration of cytoplasmic ionized calcium, [Ca²⁺]i, was measured in monolayers of fused myotubes using the Ca²⁺ indicator indo-1. The contributions of independent routes of Ca²⁺ influx and efflux to/from the cytoplasm on [Ca²⁺]i were investigated. The resting [Ca²⁺]i was 170–190 nM in different cell clones. Acetylcholine increased [Ca²⁺]i by about 2-fold in the presence or absence of extracellular Ca²⁺. Cell depolarization by K⁺ elevated [Ca²⁺]i about 3-fold, and this increase was largely dependent on extracellular Ca²⁺. Replacing Na⁺ by N-methylglucammonium⁺ raised [Ca²⁺]i > 5-fold, and 50% of this increase was dependent on extracellular Ca²⁺. All these increases in [Ca²⁺]i were transient, returning to basal [Ca²⁺]i within 2 min. It is concluded that cells in culture [Ca²⁺]i can be elevated transiently by acetylcholine through Ca²⁺ release from intracellular stores, and by K through Ca²⁺ influx. The return to basal [Ca²⁺]i is due to Na⁺/Ca²⁺ exchange and Ca²⁺-ATPase activity.

References (21)

J.D. David et al.
Dev. Biol
(1981)
A.H. Caswell et al.
Trends Bioch. Sci
(1989)
N.R. Brandt et al.
J. Biol. Chem
(1980)
C.J. Brandl et al.
Cell
(1986)
A. Klip et al.
Arch. Biochem. Biophys
(1986)
R. Yasin et al.
J. Neurol. Sci
(1977)
G. Grynkiewicz et al.
J. Biol. Chem
(1985)
A. Klip et al.
J. Biol. Chem
(1987)
T. Kitazawa et al.
J. Biol. Chem
(1989)
R.W. Tsien
Annu. Rev. Physiol
(1983)

There are more references available in the full text version of this article.

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A human intravenous immunoglobulin preparation (IVIg) released Ca²⁺ from the sarcoplasmic reticulum of cultured human skeletal muscle cells in a dose-dependent manner. Blocking the dihydropyridine–ryanodine receptor complex abrogated the IVIg-mediated Ca²⁺ response, whereas inhibition of the voltage-operated Na⁺-channels or acetylcholine receptors did not. This effect of IVIg was not mediated by its main component, the IgG molecules, and differed between preparations from different manufacturers. Heating destroyed the activity. Data shows that an unidentified serum protein present in IVIg can influence human muscle cells by an effect on the dihydropyridine receptor. This phenomenon may be important in interpreting the (side) effects of IVIg in neuromuscular diseases.
Excitation-contraction coupling of cultured human skeletal muscle cells and the relation between basal cytosolic Ca<sup>2+</sup> and excitability
1997, Cell Calcium
Cultured human skeletal muscle cells are frequently used as a model to study muscle pathology, in which Ca²⁺ homeostasis might be affected. However, their excitation-contraction (E-C) coupling has been poorly investigated.
In order to elucidate E-C coupling of cultured muscle cells, we activated the acetylcholine receptors, voltage-dependent Na⁺ channels, dihydropyridine receptors or ryanodine receptors both in the presence and absence of external Ca²⁺, as well as after specific inhibition, and measured the effects on the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) using Fura-2. Furthermore, we examined the excitability of these cells during iterative high (125 mM) K⁺ stimulation with various repolarisation intervals.
The resting [Ca²⁺]_i in muscle cells of controls is about 130 nM. Acetylcholine, veratridine, high K⁺ and caffeine elicit dose-dependent Ca²⁺ transients, which are independent of extracellular Ca²⁺ and can be inhibited by α-bungarotoxin, tetrodotoxin, nifedipine or ryanodine.
During repetitive K⁺ stimulation, the excitability of the muscle cells depends on the repolarisation interval between successive stimulations. Upon shortening the repolarisation time the Ca²⁺ transients become smaller and slower. Thereby, the basal [Ca²⁺]_i rises, the Ca²⁺ response amplitude declines and both the half-increase and half-decay time increase. However, if the basal [Ca²⁺]_i equals the resting [Ca²⁺]_i, the initial Ca²⁺ response can be recovered. The intracellular pH of 7.23, measured by BCECF, is unaffected by repeated K⁺ stimulation, whatever the repolarisation interval was.
In conclusion, cultured human skeletal muscle cells possess a ‘skeletal muscle type’ of E-C coupling and their excitability at iterative stimulation is set by their basal [Ca²⁺]_i.
Measurement of membrane potential and myoplasmic [Ca<sup>2+</sup>] in developing rat myotubes at rest and in response to stimulation
1996, Cell Calcium
In this study, the membrane potential and cytosolic [Ca²⁺] were measured in rat myotubes developing in culture from days 6–14. It was found that as the myotubes developed in culture, the resting membrane potential (RMP) became more negative during days 6–8, and then did not significantly change until after day 13, when it started to become less negative. The mean RMP measured at days 8–13 was −59 ± 1 mV (n = 70). The amplitude of action potentials elicited in the myotubes by anode break stimulation increased in size during development (range: 47.5–119 mV) and this closely correlated with the development of a more negative RMP. Cytosolic [Ca²⁺] was measured in the rat myotubes using the Ca ²⁺ indicator Fura-2, and no significant change in the resting [Ca²⁺] was observed during development (days 6–14). Ca²⁺ responses triggered by action potentials varied from small slow increases in [Ca²⁺] that failed to return to the baseline to rapid [Ca²⁺] transients. The size of the [Ca²⁺] transients positively correlated with both the observed increase in the RMP during development and the size of the action potential. Larger [Ca²⁺] transients also had more rapid rates of [Ca²⁺] decay, indicating a tandem increase in the ability of the sarcoplasmic reticulum to release and resequester Ca²⁺ during development of rat myotubes. Repetitive stimulation (10 Hz) of the myotubes exhibiting small [Ca²⁺] transients produced a step-like rise in [Ca²⁺]. Many myotubes exhibiting larger [Ca²⁺] transients could not be stimulated at 10 Hz by anode break stimulation due to the presence of action potentials with large hyperpolarisations. However, when these myotubes were depolarised at 10 Hz, they produced a tetanic Ca²⁺ response similar to that seen in adult skeletal muscle.
Changes in cytosolic resting ionized calcium level and in calcium transients during in vitro development of normal and Duchenne muscular dystrophy cultured skeletal muscle measured by laser cytofluorimetry using Indo-1
1993, Cell Calcium
Intracellular calcium activity was recorded during in vitro myogenesis of human normal and DMD muscle, using the calcium probe Indo-1 under laser illumination, at rest and during different kinds of stimulation (acetylcholine, high K⁺, caffeine). In myoblasts, the resting intracellular calcium level was significantly larger in DMD cells (89 ± 9 nM; n = 40 vs 37 ± 5 nM; n = 22) but there was no significant difference in myotubes, after fusion (44 ± 4 nM; n = 34 vs 36 ± 4 nM; n = 52). A similar evolution was observed in cells cultured from FSH biopsies. The amplitude of ACh- and high K⁺-induced calcium transients was significantly halved in DMD myotubes as compared to control ones and non-significantly decreased for caffeine responses. Some alterations in the kinetics of responses were observed in DMD muscle: the rising phases of ACh- and high K⁺-elicited transients and the decaying phase of the ACh-responses were significantly slowed down. It is concluded that: (i) in aneurally cultured human muscle, an increase in the basal level of internal calcium can occur at early stages of myogenesis before the expression of the dystrophin gene; and (ii) the changes in calcium transients induced by depolarization or direct stimulation of sarcoplasmic reticulum are not susceptible of inducing a calcium overload in DMD cells.
Intracellular calcium transients induced by different kinds of stimulus during myogenesis of rat skeletal muscle cells studied by laser cytofluorimetry with Indo-1
1993, Cell Calcium
Resting intracellular calcium levels and intracellular calcium transients induced by three types of stimulus (acetylcholine, high potassium and caffeine) were recorded, during in vitro myogenesis, by means of a ratiometric fluorescence method using the calcium probe Indo-1 under laser illumination. Resting levels seemed to decrease with the age of cultured cells and the depolarization-induced transients, through 100 mM K⁺ or Ach application, were progressively faster and larger as the muscle cells developed. An additive mechanism, likely due to calcium entry into the cell through nicotinic acetylcholine receptors, could explain the differences observed in Ach-induced responses as compared with the 100 mM K⁺-induced ones. In myoballs (the older cells) the calcium transients exhibited progressively a biphasic shape. From data obtained in different conditions (tetrodotoxin, nifedipine, strontium and free Ca EGTA) and those indicating the appearance of caffeine-releasable intracellular calcium stores only at 2–3 days stage, and from the previously reported developmental appearance of calcium currents and contraction, it was proposed that, in young myotubes, the calcium transients were more dependent on extracellular calcium than in older cells. These developmental data are discussed in the light of a known model of the in situ biogenesis of the structures involved in excitation-contraction coupling (ECC) like transverse tubules and triads.
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1991, Biochemical and Biophysical Research Communications
Skeletal muscle is the main tissue responsible for glucose utilization in the fed state, and it expresses the ubiquitous GLUT1 glucose transporter and the muscle/fat specific GLUT4 glucose transporter. Here we investigated the expression of these transporters during muscle cell differentiation in vitro. Rat L6 muscle cells were grown to the stages of myoblasts, alignment and fused myotubes. Glucose (2-deoxy-D-glucose) transport was higher in myoblasts, decreasing with the progression of alignment and cell fusion. Conversely, insulin-stimulated glucose uptake was negligible in myoblasts, and increased with cell alignment and fusion. The cellular content of GLUT1 transporters decreased and that of GLUT4 transporters increased with cell fusion. Insulin rapidly stimulated glucose uptake in fused myotubes maintained in 2% serum but not in 10% serum. In 10% serum, basal glucose uptake increased as did the cellular content of GLUT1 transporters, while GLUT4 transporter content did not change. These results indicate that both transporters are regulated oppositely during muscle cell differentiation, and that high serum concentrations override the capacity of insulin to regulate transport by inducing overexpression of the GLUT1 transporter.

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Regulation of cytosolic Ca2+ in clonal human muscle cell cultures

Abstract

Dev. Biol

Trends Bioch. Sci

J. Biol. Chem

Cell

Arch. Biochem. Biophys

J. Neurol. Sci

J. Biol. Chem

J. Biol. Chem

J. Biol. Chem

Annu. Rev. Physiol

Regulation of cytosolic Ca²⁺ in clonal human muscle cell cultures