scholarly journals A carnitine/acylcarnitine translocase assay applicable to biopsied muscle specimens without requiring mitochondrial isolation

1986 ◽  
Vol 236 (1) ◽  
pp. 143-148 ◽  
Author(s):  
M S R Murthy ◽  
V S Kamanna ◽  
S V Pande
Keyword(s):  
Skeletal Muscle ◽  
Rat Heart ◽  
Skeletal Muscles ◽  
Human Skeletal Muscle ◽  
Carnitine Acetyltransferase ◽  
Fresh Tissue ◽  
Simple Method ◽  
Oxidase System ◽  
Acylcarnitine Translocase ◽  
Mitochondrial Transporters

A simple method for assaying the mitochondrial carnitine/acylcarnitine translocase of muscles that needs only few milligrams of fresh tissue is described. The procedure involves monitoring of the sulphobetaine (an inhibitor of the translocase)-sensitive acetylation of sub-saturating concentrations of carnitine in the medium, linked to the oxidation of [2-14C]pyruvate in the presence of malonate. Conditions affecting the reliability of the outlined procedure and the ancillary information to be collected, namely the activities of pyruvate oxidase system and carnitine acetyltransferase, for detecting possible deficiency of the translocase are described, together with data on the translocase activity in human skeletal muscle, in rat red and white skeletal muscles and in rat heart. The concepts outlined should allow development of assays of other mitochondrial transporters that also would require neither isolation of mitochondria nor availability of a large quantity of tissue, both of which are otherwise needed at present.

scholarly journals Isolation of human skeletal muscle myosin heavy chain and actin for measurement of fractional synthesis rates

1998 ◽  
Vol 275 (6) ◽  
pp. E1092-E1099 ◽  
Author(s):  
D. L. Hasten ◽  
G. S. Morris ◽  
S. Ramanadham ◽  
K. E. Yarasheski
Keyword(s):  
Skeletal Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Human Skeletal Muscle ◽  
Sodium Dodecyl ◽  
Synthesis Rate ◽  
Simple Method ◽  
Skeletal Muscle Myosin ◽  
Fractional Synthesis

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we have developed a simple method to isolate myosin heavy chain (MHC) and actin from small (60–80 mg) human skeletal muscle samples for the determination of their fractional synthesis rates. The amounts of MHC and actin isolated are adequate for the quantification of [13C]leucine abundance by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Fractional synthesis rates of mixed muscle protein (MMP), MHC, and actin were determined in six healthy young subjects (27 ± 1 yr) after they received a 14-h intravenous infusion (prime = 7.58 μmol/kg body wt, constant infusion = 7.58 μmol ⋅ kg body wt−1 ⋅ h−1) of [1-13C]leucine. The fractional synthesis rates of MMP, MHC, and actin were found to be 0.0468 ± 0.0048, 0.0376 ± 0.0033, and 0.0754 ± 0.0078%/h, respectively. Overall, the synthesis rate of MHC was 20% lower ( P = 0.012), and the synthesis rate of actin was 61% higher ( P = 0.060, not significant) than the MMP synthesis rate. The isolation of these proteins for isotope abundance analysis by GC-C-IRMS provides important information about the synthesis rates of these specific contractile proteins, as opposed to the more general information provided by the determination of MMP synthesis rates.

scholarly journals A 3D culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mohsen Afshar Bakooshli ◽  
Ethan S Lippmann ◽  
Ben Mulcahy ◽  
Nisha Iyer ◽  
Christine T Nguyen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Human Skeletal Muscle ◽  
De Novo ◽  
Muscle Fibers ◽  
3D Culture ◽  
Simple Method ◽  
Adult Human

Two-dimensional (2D) human skeletal muscle fiber cultures are ill-equipped to support the contractile properties of maturing muscle fibers. This limits their application to the study of adult human neuromuscular junction (NMJ) development, a process requiring maturation of muscle fibers in the presence of motor neuron endplates. Here we describe a three-dimensional (3D) co-culture method whereby human muscle progenitors mixed with human pluripotent stem cell-derived motor neurons self-organize to form functional NMJ connections. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-cultures. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. Hence, this work offers a simple method to model and evaluate adult human NMJ de novo development or disease in culture.

Proteomic identification of secreted proteins from human skeletal muscle cells and expression in response to strength training

2011 ◽  
Vol 301 (5) ◽  
pp. E1013-E1021 ◽  
Author(s):  
Frode Norheim ◽  
Truls Raastad ◽  
Bernd Thiede ◽  
Arild C. Rustan ◽  
Christian A. Drevon ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Strength Training ◽  
Skeletal Muscles ◽  
Human Skeletal Muscle ◽  
Vastus Lateralis ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Secreted Proteins ◽  
Human Myotubes ◽  
Human Skeletal Muscle Cells

Regular physical activity protects against several types of diseases. This may involve altered secretion of signaling proteins from skeletal muscle. Our aim was to identify the most abundantly secreted proteins in cultures of human skeletal muscle cells and to monitor their expression in muscles of strength-training individuals. A total of 236 proteins were detected by proteome analysis in medium conditioned by cultured human myotubes, which was narrowed down to identification of 18 classically secreted proteins expressed in skeletal muscle, using the SignalP 3.0 and Human Genome Expression Profile databases together with a published mRNA-based reconstruction of the human skeletal muscle secretome. For 17 of the secreted proteins, expression was confirmed at the mRNA level in cultured human myotubes as well as in biopsies of human skeletal muscles. RT-PCR analyses showed that 15 of the secreted muscle proteins had significantly enhanced mRNA expression in m. vastus lateralis and/or m. trapezius after 11 wk of strength training among healthy volunteers. For example, secreted protein acidic and rich in cysteine, a secretory protein in the membrane fraction of skeletal muscle fibers, was increased 3- and 10-fold in m. vastus lateralis and m. trapezius, respectively. Identification of proteins secreted by skeletal muscle cells in vitro facilitated the discovery of novel responses in skeletal muscles of strength-training individuals.

Effects of delayed freezing on content of phosphagens in human skeletal muscle biopsy samples

1986 ◽  
Vol 61 (3) ◽  
pp. 832-835 ◽  
Author(s):  
K. Soderlund ◽  
E. Hultman
Keyword(s):  
Skeletal Muscle ◽  
Muscle Biopsy ◽  
Human Skeletal Muscle ◽  
Lactate Concentration ◽  
Energy Utilization ◽  
Gas Mixture ◽  
Rapid Freezing ◽  
Atp Content ◽  
Fresh Tissue ◽  
Skeletal Muscle Biopsy

The concentrations of ATP, phosphocreatine (PCr), creatine, and lactate were determined in muscle biopsy samples frozen immediately or after a delay of 1–6 min. During the delay the samples were exposed to normal air or a gas mixture of 6.5% CO2-93.5% O2. The ATP content was unchanged, but PCr increased significantly from 72 mmol after rapid freezing to 85 mmol X kg dry muscle-1 during the 1st min in air. The lactate concentration increased (2.8 to 5.2 mmol X kg-1). If muscles were made anoxic by circulatory occlusion for 4–6 min before sampling, no increase in PCr was observed. Direct homogenization of fresh tissue in perchloric acid gave the same ATP, PCr, and lactate contents as frozen samples. It is concluded that the ATP and PCr contents in muscle are unaffected by freezing but that the biopsy procedure activates the energy utilization processes resulting in PCr decrease. It is suggested that the muscle PCr content after a 1-min delay in tissue freezing corresponds to the level in resting fresh muscle.

Inhibition of mitochondrial carnitine–acylcarnitine translocase by sulfobetaines

1980 ◽  
Vol 58 (10) ◽  
pp. 822-830 ◽  
Author(s):  
Rehana Parvin ◽  
Tapas Goswami ◽  
Shri V. Pande
Keyword(s):  
Rat Heart ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Carnitine Acetyltransferase ◽  
Selective Inhibitors ◽  
Maximal Inhibition ◽  
Isolation Medium ◽  
Carnitine Acyltransferases ◽  
Rat Heart Mitochondria ◽  
Acylcarnitine Translocase

Sulfobetaines (N-alkyl-N,N-dimethyl-3-ammonio-1-propanesulfonates) have been identified as relatively specific and selective inhibitors of mitochondrial carnitine–acylcarnitine translocase. Thus, sublytic concentrations of sulfobetaines (alkyl = octyl to tetradecyl) inhibit the respiration of rat heart mitochondria supported by added acylcarnitines or pyruvate plus malonate and carnitine. Both exchange efflux and unidirectional net efflux of mitochondrial carnitine are also inhibited; the half-maximal inhibition of the former occurs at micromolar concentrations of sulfobetaines and the inhibitory effect is reversible and competitive with respect to carnitine. As a stop-inhibitor, 20 mM sulfobetaine8, (alkyl = octyl), is useable at near 0 °C but is less effective than 2 mM mersalyl when transport rates are very rapid as at higher temperatures especially with liver mitochondria. The loss of mitochondrial carnitine that normally occurs owing to the progress of net efflux during the isolation of mitochondria is prevented by the inclusion of 20 mM sulfobetaine8 in the isolation medium and this enables a better estimate of the mitochondrial carnitine content. Sulfobetaines inhibit the activities of mitochondrial carnitine acetyltransferase and carnitine palmitoyltransferase but only at concentrations severalfold higher than those inhibitory for the translocase. This observation supports the belief that carnitine–acylcarnitine translocase is an entity distinct from that of carnitine acyltransferases.

The Use of Human Skeletal Muscle in Vitro for Biochemical and Pharmacological Studies of Glucose Uptake

1973 ◽  
Vol 44 (1) ◽  
pp. 55-62 ◽  
Author(s):  
K. N. Frayn ◽  
P. I. Adnitt ◽  
P. Turner
Keyword(s):  
Skeletal Muscle ◽  
Glucose Metabolism ◽  
Glucose Uptake ◽  
Metabolic Control ◽  
Rat Heart ◽  
Human Skeletal Muscle ◽  
Antidiabetic Drug ◽  
Pharmacological Studies

1. A method is described for measuring glucose uptake by pieces of human skeletal muscle removed at surgery. 2. Glucose uptake by this preparation was stimulated by insulin (0.1–10 munits/ml) and inhibited by 2.27 mM-sodium n-butyrate in the presence of insulin (0.1 munit/ml). 3. The antidiabetic drug metformin (60.4 μM) had no effect on glucose uptake in the absence or presence of insulin (0.1 munit/ml), but stimulated uptake in the presence of 2.27 mM-sodium n-butyrate together with insulin (0.1 munit/ml). 4. It is concluded that the metabolic control of glucose uptake in human skeletal muscle is similar to that in rat heart and diaphragm, and that the preparation provides a useful model for biochemical and pharmacological investigations of glucose metabolism in skeletal muscle.

scholarly journals Gene expression effects of glucocorticoid and mineralocorticoid receptor agonists and antagonists on normal human skeletal muscle

2017 ◽  
Vol 49 (6) ◽  
pp. 277-286 ◽  
Author(s):  
Jessica A. Chadwick ◽  
J. Spencer Hauck ◽  
Celso E. Gomez-Sanchez ◽  
Elise P. Gomez-Sanchez ◽  
Jill A. Rafael-Fortney
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Skeletal Muscles ◽  
Human Skeletal Muscle ◽  
Expression Profiles ◽  
Combined Treatment ◽  
Gene Expression Profiles ◽  
Steroid Hormone Receptors

Mineralocorticoid and glucocorticoid receptors are closely related steroid hormone receptors that regulate gene expression through many of the same hormone response elements. However, their transcriptional activities and effects in skeletal muscles are largely unknown. We recently identified mineralocorticoid receptors (MR) in skeletal muscles after finding that combined treatment with the angiotensin-converting enzyme inhibitor lisinopril and MR antagonist spironolactone was therapeutic in Duchenne muscular dystrophy mouse models. The glucocorticoid receptor (GR) agonist prednisolone is the current standard-of-care treatment for Duchenne muscular dystrophy because it prolongs ambulation, likely due to its anti-inflammatory effects. However, data on whether glucocorticoids have a beneficial or detrimental direct effect on skeletal muscle are controversial. Here, we begin to define the gene expression profiles in normal differentiated human skeletal muscle myotubes treated with MR and GR agonists and antagonists. The MR agonist aldosterone and GR agonist prednisolone had highly overlapping gene expression profiles, supporting the notion that prednisolone acts as both a GR and MR agonist that may have detrimental effects on skeletal muscles. Co-incubations with aldosterone plus either nonspecific or selective MR antagonists, spironolactone or eplerenone, resulted in similar numbers of gene expression changes, suggesting that both drugs can block MR activation to a similar extent. Eplerenone treatment alone decreased a number of important muscle-specific genes. This information may be used to develop biomarkers to monitor clinical efficacy of MR antagonists or GR agonists in muscular dystrophy, develop a temporally coordinated treatment with both drugs, or identify novel therapeutics with more specific downstream targets.

TGFß regulates metabolism of human skeletal muscle cells by miRNAs

2018 ◽  
Author(s):  
S Höckele ◽  
P Huypens ◽  
C Hoffmann ◽  
T Jeske ◽  
M Hastreiter ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Human Skeletal Muscle ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Human Skeletal Muscle Cells

IRAK-1/IRS-1 Signaling Cross Talk in Human Skeletal Muscle—Role in Insulin Resistance

Diabetes ◽  
2018 ◽  
Vol 67 (Supplement 1) ◽  
pp. 159-OR
Author(s):  
THEODORE P. CIARALDI ◽  
SUNDER MUDALIAR ◽  
LIWU LI ◽  
ROSARIO SCALIA ◽  
XIAO JIAN SUN ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Cross Talk ◽  
Human Skeletal Muscle
Sign in / Sign up

Export Citation Format

Share Document