Quantitative study of motor endplates in muscle fibres dissociated by a simple procedure

Large numbers of single muscle fibres can be obtained reproducibly from glutaraldehyde-fixed skeletal muscle by the method described here. With suitable modifications, one can estimate acetylcholine receptor number (α-bungarotoxin binding sites) and endplate area in parallel portions produced from the same muscle sample, so that small differences (e. g. with growth or between muscle types) become detectable. Micro-dissection further increases the precision of evaluation of junctional, perijunctional and extrajunctional binding sites. Other applications are illustrated.

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Interaction of di-iodinated 125I-labelled α-bungarotoxin and reversible cholinergic ligands with intact synaptic acetylcholine receptors on isolated skeletal-muscle fibres from the rat

Biochemical Journal ◽

10.1042/bj1810545 ◽

1979 ◽

Vol 181 (3) ◽

pp. 545-557 ◽

Cited By ~ 8

Author(s):

P Darveniza ◽

J A Morgan-Hughes ◽

E J Thompson

Keyword(s):

Skeletal Muscle ◽

Dissociation Constant ◽

Binding Sites ◽

Acetylcholine Receptors ◽

Time Course ◽

Muscle Fibres ◽

First Order ◽

Toxin Binding ◽

Alpha Bungarotoxin ◽

Skeletal Muscle Fibres

1. Intact synaptic acetylcholine receptors on freshly isolated rat skeletal-muscle fibres were characterized by their interaction with di-iodinated 125I-labelled alpha-bungarotoxin, acetylcholine and other cholinergic ligands at room temperature (22 deggrees C). 2. The time course and concentration dependence of 125I-labelled alpha-bungarotoxin association conformed to a bimolecular mechanism. In time-course experiments with different concentrations of 125I-labelled alpha-bungarotoxin (1.4–200 nM) the bimolecular-association rate constant, k + 1, was (2.27 +/- 0.49) × 10(4)M-1.S-1 (mean +/- S.D., N = 10). In concentration-dependence experiments, k + 1 was 2.10 × 10(4)M-1.S-1 and 1.74 × 10(4) M-1.S-1 with 10 and 135 min incubations respectively. In association experiments the first-order rate constant was proportional to the 125I-labelled alpha-bungarotoxin concentration. 125I-Labelled alpha-bungarotoxin dissociation was first order with a dissociation constant, k-1, less than or equal to 3 × 10(-6)S(-1) (half-life greater than or equal to 60 h.) The results indicated a single class of high-affinity toxin-binding sites at the end-plate with an equilibrium dissociation constant, Kd, equal to or less than 100 pM. The number of toxin-binding sites was (3.62 +/- 0.46) × 10(7) (mean +/- S.D., n = 22) per rat end-plate. 3. The apparent inhibitor dissociation constants, Ki, for reversible cholinergic ligands were determined by studying their effect at equilibrium on the rate of 125I-labelled alpha-bungarotoxin binding. There was heterogeneity of binding sites for cholinergic ligands, which were independent and non-interacting with antagonists. In contrast agonist affinity decreased with increasing receptor occupancy. Cholinergic ligands in excess inhibited over 90% of 125I-labelled alpha-bungarotoxin binding. 4. Cholinergic ligand binding was accompanied by an increase in entropy, which was greater for the agonist carbachol (delta So = +0.46 kJ.mol-1.K-1) than the antagonist tubocurarine (delta So = +0.26 kJ.mol-1.K-1). 5. The entropy and affinity changes that accompanied agonist binding suggested that agonists induced significant conformational changes in intact acetylcholine receptors. 6. The affinity and specificity of 125I-labelled alpha-bungarotoxin and tubocurarine binding to synaptic acetylcholine receptors from slow and fast muscle fibres were the same. 7. The study of binding only requires milligram amounts of tissue and may have application to other neurobiological studies and to the study of human neuromuscular disorders.

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Pharmacological distinction between dantrolene and ryanodine binding sites: evidence from normal and malignant hyperthermia-susceptible porcine skeletal muscle

Biochemical Journal ◽

10.1042/bj3260847 ◽

1997 ◽

Vol 326 (3) ◽

pp. 847-852 ◽

Cited By ~ 29

Author(s):

Sanjay S. PALNITKAR ◽

James R. MICKELSON ◽

Charles F. LOUIS ◽

Jerome PARNESS

Keyword(s):

Skeletal Muscle ◽

Malignant Hyperthermia ◽

Binding Sites ◽

Calcium Release ◽

Drug Binding ◽

Protein Patterns ◽

Open Probability ◽

Malignant Hyperthermia Susceptible ◽

Discontinuous Sucrose Gradient ◽

Muscle Types

Dantrolene inhibits and ryanodine stimulates calcium release from skeletal-muscle sarcoplasmic reticulum (SR), the former by an unknown mechanism, and the latter by activating the ryanodine receptor (RyR), the primary Ca2+-release channel of SR. Dantrolene is used to treat malignant hyperthermia (MH), a genetic predisposition to excessive intracellular Ca2+ release upon exposure to volatile anaesthetics. Porcine MH results from a point mutation in the SR RyR that alters the open probability of the channel, and is reflected in altered [3H]ryanodine binding parameters. Specific binding sites for [3H]dantrolene and [3H]ryanodine co-distribute on SR that has been isolated by discontinuous sucrose gradient centrifugation. If the two drug-binding sites are functionally linked, [3H]dantrolene binding might be affected both by pharmacological and by genetic modulators of the functional state of the RyR. Accordingly, we compared the characteristics of [3H]dantrolene binding to porcine malignant-hyperthermia-susceptible and normal-skeletal-muscle SR, and examined the effects of RyR modulators on [3H]dantrolene binding to these membranes. Additionally, the feasibility of separating the SR binding sites for [3H]dantrolene and [3H]ryanodine was investigated. No significant differences in [3H]dantrolene binding characteristics to SR membranes from the two muscle types were detected, and the Bmax ratio for [3H]dantrolene/[3H]ryanodine was 1.4(±0.1):1 in both muscle types. [3H]Dantrolene binding is unaffected by the RyR modulators caffeine, ryanodine, Ruthenium Red and calmodulin, and neither dantrolene nor azumolene have any effect on [3H]ryanodine binding. Additionally, distinct peaks of [3H]dantrolene and [3H]ryanodine binding are detected in SR membranes fractionated by linear sucrose centrifugation, although no differences in protein patterns are detected by SDS/PAGE or Western-blot analysis. We suggest that the binding sites for these two drugs are pharmacologically distinct, and may exist on separate molecules.

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A single muscle contusion promotes an immediate alteration in mitochondrial bioenergetics response in skeletal muscle fibres with different metabolism

Free Radical Research ◽

10.1080/10715762.2020.1723795 ◽

2020 ◽

Vol 54 (2-3) ◽

pp. 137-149

Author(s):

Diane D. Hartmann ◽

Débora F. Gonçalves ◽

Pamela C. Da Rosa ◽

Rodrigo P. Martins ◽

Aline A. Courtes ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Fibres ◽

Mitochondrial Bioenergetics ◽

Single Muscle ◽

Skeletal Muscle Fibres

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Decoding mitochondrial heterogeneity in single muscle fibres by imaging mass cytometry

Scientific Reports ◽

10.1038/s41598-020-70885-3 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Charlotte Warren ◽

David McDonald ◽

Roderick Capaldi ◽

David Deehan ◽

Robert W. Taylor ◽

...

Keyword(s):

Skeletal Muscle ◽

Oxidative Phosphorylation ◽

Single Cell ◽

Mitochondrial Disease ◽

Muscle Fibres ◽

Single Muscle ◽

Single Cell Level ◽

Mass Cytometry ◽

Cell Level ◽

Wide Range

Abstract The study of skeletal muscle continues to support the accurate diagnosis of mitochondrial disease and remains important in delineating molecular disease mechanisms. The heterogeneous expression of oxidative phosphorylation proteins and resulting respiratory deficiency are both characteristic findings in mitochondrial disease, hence the rigorous assessment of these at a single cell level is incredibly powerful. Currently, the number of proteins that can be assessed in individual fibres from a single section by immunohistochemistry is limited but imaging mass cytometry (IMC) enables the quantification of further, discrete proteins in individual cells. We have developed a novel workflow and bespoke analysis for applying IMC in skeletal muscle biopsies from patients with genetically-characterised mitochondrial disease, investigating the distribution of nine mitochondrial proteins in thousands of single muscle fibres. Using a semi-automated analysis pipeline, we demonstrate the accurate quantification of protein levels using IMC, providing an accurate measure of oxidative phosphorylation deficiency for complexes I–V at the single cell level. We demonstrate signatures of oxidative phosphorylation deficiency for common mtDNA variants and nuclear-encoded complex I variants and a compensatory upregulation of unaffected oxidative phosphorylation components. This technique can now be universally applied to evaluate a wide range of skeletal muscle disorders and protein targets.

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Electron Probe Analysis of Muscle

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100054583 ◽

1982 ◽

Vol 40 ◽

pp. 398-401

Author(s):

A. V. Somlyo ◽

H. Shuman ◽

A. P. Somlyo

Keyword(s):

Skeletal Muscle ◽

Smooth Muscle ◽

Sarcoplasmic Reticulum ◽

Resting State ◽

Binding Sites ◽

Electron Probe ◽

Frog Skeletal Muscle ◽

Electron Probe Analysis ◽

Probe Analysis

Electron probe analysis of frozen dried cryosections of frog skeletal muscle, rabbit vascular smooth muscle and of isolated, hyperpermeab1 e rabbit cardiac myocytes has been used to determine the composition of the cytoplasm and organelles in the resting state as well as during contraction. The concentration of elements within the organelles reflects the permeabilities of the organelle membranes to the cytoplasmic ions as well as binding sites. The measurements of [Ca] in the sarcoplasmic reticulum (SR) and mitochondria at rest and during contraction, have direct bearing on their role as release and/or storage sites for Ca in situ.

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