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.

scholarly journals A three-dimensional culture model of innervated human skeletal muscle enables studies of the adult neuromuscular junction and disease modeling

2018 ◽  
Author(s):  
Mohsen Afshar Bakooshli ◽  
Ethan S Lippmann ◽  
Ben Mulcahy ◽  
Nisha R Iyer ◽  
Christine T Nguyen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Human Skeletal Muscle ◽  
Disease Modeling ◽  
De Novo ◽  
Muscle Fibers ◽  
Three Dimensional ◽  
Adult Human

SummaryTwo-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 within two weeks. Functional connectivity between motor neuron endplates and muscle fibers is confirmed with calcium transient imaging and electrophysiological recordings. Notably, we only observed epsilon acetylcholine receptor subunit protein upregulation and activity in 3D co-culture. This demonstrates that the 3D co-culture system supports a developmental shift from the embryonic to adult form of the receptor that does not occur in 2D co-culture. Further, 3D co-culture treatments with myasthenia gravis patient sera shows the ease of studying human disease with the system. This work delivers a simple, reproducible, and adaptable method to model and evaluate adult human NMJ de novo development and disease in culture.

Induction of GLUT-1 protein in adult human skeletal muscle fibers

2000 ◽  
Vol 279 (5) ◽  
pp. E1191-E1195 ◽  
Author(s):  
M. Gaster ◽  
J. Franch ◽  
P. Staehr ◽  
H. Beck-Nielsen ◽  
T. Smith ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Human Skeletal Muscle ◽  
Contractile Activity ◽  
Muscle Fibers ◽  
Insulin Dependent Diabetes Mellitus ◽  
Human Muscle ◽  
Dependent Diabetes Mellitus ◽  
Glut 1 ◽  
Adult Human ◽  
Skeletal Muscle Fibers

Prompted by our recent observations that GLUT-1 is expressed in fetal muscles, but not in adult muscle fibers, we decided to investigate whether GLUT-1 expression could be reactivated. We studied different stimuli concerning their ability to induce GLUT-1 expression in mature human skeletal muscle fibers. Metabolic stress (obesity, non-insulin-dependent diabetes mellitus), contractile activity (training), and conditions of de- and reinnervation (amyotrophic lateral sclerosis) could not induce GLUT-1 expression in human muscle fibers. However, regenerating muscle fibers in polymyositis expressed GLUT-1. In contrast to GLUT-1, GLUT-4 was expressed in all investigated muscle fibers. Although the significance of GLUT-1 in adult human muscle fibers appears limited, GLUT-1 may be of importance for the glucose supplies in immature and regenerating muscle.

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

2019 ◽  
Author(s):  
Mohsen Afshar Bakooshli ◽  
Ethan S Lippmann ◽  
Ben Mulcahy ◽  
Nisha Iyer ◽  
Christine T Nguyen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Neuromuscular Junction ◽  
Human Skeletal Muscle ◽  
3D Culture ◽  
Author Response ◽  
Culture Model ◽  
3D Culture Model

scholarly journals Immunolocalization of AMP-deaminase isozymes in human skeletal muscle and cultured muscle cells: concentration of isoform M at the neuromuscular junction.

1994 ◽  
Vol 42 (7) ◽  
pp. 861-868 ◽  
Author(s):  
T H van Kuppevelt ◽  
J H Veerkamp ◽  
W N Fishbein ◽  
N Ogasawara ◽  
R L Sabina
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Neuromuscular Junction ◽  
Smooth Muscle Cells ◽  
Human Skeletal Muscle ◽  
Muscle Fibers ◽  
Muscle Cells ◽  
Amp Deaminase ◽  
Nerve Bundles ◽  
Cross Striation

The three major isoforms of AMP-deaminase (AMPda) were localized in human skeletal muscle and cultured muscle cells by immunocytochemistry. The M isoform was mainly located in Type II muscle fibers and showed a clear cross-striation. Particularly strong staining was present at the neuromuscular junction. Capillaries were also immunoreactive. The L isoform was predominantly observed in nerve bundles and to a minor extent in smooth muscle cells and endothelial cells. The E isoform was predominantly present in smooth muscle cells, and to a lesser extent in Type I muscle fibers and nerve bundles. In quadriceps muscle of patients with myoadenylate deaminase deficiency, no immunostaining for the M isozyme was observed, whereas reactivity for the L and E isoforms was unaltered. In human muscle cell cultures, mononuclear cells, including myoblasts, were immunoreactive for the L isoform and to a lesser extent the E isoform, whereas the M isoform was absent. In myotubes, diffuse or fibrillar staining was present for all three isoforms, but only the M isoform showed a clear cross-striation pattern in highly differentiated myotubes.

scholarly journals Development of a human neuromuscular tissue-on-a-chip model on a 24-well-plate-format compartmentalized microfluidic device

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Kazuki Yamamoto ◽  
Nao Yamaoka ◽  
Yu Imaizumi ◽  
Takunori Nagashima ◽  
Taiki Furutani ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Microfluidic Device ◽  
Motor Neurons ◽  
Muscle Fibers ◽  
Three Dimensional ◽  
Tissue Model ◽  
Skeletal Muscle Fibers

A three-dimensional human neuromuscular tissue model that mimics the physically separated structures of motor neurons and skeletal muscle fibers is presented.

scholarly journals Skeletal Muscle Metabolism: Origin or Prognostic Factor for Amyotrophic Lateral Sclerosis (ALS) Development?

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1449
Author(s):  
Cyril Quessada ◽  
Alexandra Bouscary ◽  
Frédérique René ◽  
Cristiana Valle ◽  
Alberto Ferri ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amyotrophic Lateral Sclerosis ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Muscle Metabolism ◽  
The Body ◽  
Energy Deficit ◽  
Acid Oxidation ◽  
Progression Of Disease ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive and selective loss of motor neurons, amyotrophy and skeletal muscle paralysis usually leading to death due to respiratory failure. While generally considered an intrinsic motor neuron disease, data obtained in recent years, including our own, suggest that motor neuron protection is not sufficient to counter the disease. The dismantling of the neuromuscular junction is closely linked to chronic energy deficit found throughout the body. Metabolic (hypermetabolism and dyslipidemia) and mitochondrial alterations described in patients and murine models of ALS are associated with the development and progression of disease pathology and they appear long before motor neurons die. It is clear that these metabolic changes participate in the pathology of the disease. In this review, we summarize these changes seen throughout the course of the disease, and the subsequent impact of glucose–fatty acid oxidation imbalance on disease progression. We also highlight studies that show that correcting this loss of metabolic flexibility should now be considered a major goal for the treatment of ALS.

scholarly journals THE DISTRIBUTION OF LACTATE DEHYDROGENASE ISOZYMES IN HUMAN SKELETAL MUSCLE FIBERS

1964 ◽  
Vol 12 (8) ◽  
pp. 608-614 ◽  
Author(s):  
M. VAN WIJHE ◽  
M. C. BLANCHAER ◽  
S. ST. GEORGE-STUBBS
Keyword(s):  
Skeletal Muscle ◽  
Lactate Dehydrogenase ◽  
Human Skeletal Muscle ◽  
Muscle Fibers ◽  
Isozyme Pattern ◽  
Electrophoretic Separation ◽  
Lactate Dehydrogenase Isozymes ◽  
Dehydrogenase Isozyme ◽  
Lactate Dehydrogenase Isozyme ◽  
Normal Human

A study of the distribution of lactate dehydrogenase isozymes in single fibers from normal human skeletal muscle is presented. The fibers were classified into red, intermediate and white types on histochemical grounds and their lactate dehydrogenase isozyme content assessed by electrophoretic separation in veronal buffered agar. The results generally agreed with previous homogenate studies on animal skeletal muscle, in that the white fibers contained almost exclusively isozymes IV and V, whereas red fibers were rich in isozymes I, II and III, but IV and V also appeared indigenous to these fibers. The intermediate fibers had an isozyme pattern combining the features of red and white fibers. The metabolic implications of these findings are discussed.

Differential patterns of transcript accumulation during human myogenesis

1987 ◽  
Vol 7 (11) ◽  
pp. 4100-4114
Author(s):  
P Gunning ◽  
E Hardeman ◽  
R Wade ◽  
P Ponte ◽  
W Bains ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Carbonic Anhydrase ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Time Course ◽  
Human Skeletal Muscle ◽  
Transcript Accumulation ◽  
Mrna Accumulation ◽  
Adult Human ◽  
Alpha Actin

We evaluated the extent to which muscle-specific genes display identical patterns of mRNA accumulation during human myogenesis. Cloned satellite cells isolated from adult human skeletal muscle were expanded in culture, and RNA was isolated from low- and high-confluence cells and from fusing cultures over a 15-day time course. The accumulation of over 20 different transcripts was compared in these samples with that in fetal and adult human skeletal muscle. The expression of carbonic anhydrase 3, myoglobin, HSP83, and mRNAs encoding eight unknown proteins were examined in human myogenic cultures. In general, the expression of most of the mRNAs was induced after fusion to form myotubes. However, several exceptions, including carbonic anhydrase and myoglobin, showed no detectable expression in early myotubes. Comparison of all transcripts demonstrated little, if any, identity of mRNA accumulation patterns. Similar variability was also seen for mRNAs which were also expressed in nonmuscle cells. Accumulation of mRNAs encoding alpha-skeletal, alpha-cardiac, beta- and gamma-actin, total myosin heavy chain, and alpha- and beta-tubulin also displayed discordant regulation, which has important implications for sarcomere assembly. Cardiac actin was the only muscle-specific transcript that was detected in low-confluency cells and was the major alpha-actin mRNA at all times in fusing cultures. Skeletal actin was transiently induced in fusing cultures and then reduced by an order of magnitude. Total myosin heavy-chain mRNA accumulation lagged behind that of alpha-actin. Whereas beta- and gamma-actin displayed a sharp decrease after initiation of fusion and thereafter did not change, alpha- and beta-tubulin were transiently induced to a high level during the time course in culture. We conclude that each gene may have its own unique determinants of transcript accumulation and that the phenotype of a muscle may not be determined so much by which genes are active or silent but rather by the extent to which their transcript levels are modulated. Finally, we observed that patterns of transcript accumulation established within the myotube cultures were consistent with the hypothesis that myoblasts isolated from adult tissue recapitulate a myogenic developmental program. However, we also detected a transient appearance of adult skeletal muscle-specific transcripts in high-confluence myoblast cultures. This indicates that the initial differentiation of these myoblasts may reflect a more complex process than simple recapitulation of development.

RPS6 phosphorylation occurs to a greater extent in the periphery of human skeletal muscle fibers, near focal adhesions, after anabolic stimuli

2021 ◽  
Author(s):  
Nathan Hodson ◽  
Michael Mazzulla ◽  
Maksym N. H. Holowaty ◽  
Dinesh Kumbhare ◽  
Daniel R. Moore
Keyword(s):  
Skeletal Muscle ◽  
Focal Adhesion ◽  
Human Skeletal Muscle ◽  
Muscle Fibers ◽  
Focal Adhesions ◽  
Immunofluorescent Staining ◽  
Whole Body ◽  
Vastus Lateralis Muscle ◽  
Cell Periphery ◽  
Rps6 Phosphorylation

Following anabolic stimuli (mechanical loading and/or amino acid provision) the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1-mediated phosphorylation events occur in these peripheral regions or prior to translocation (i.e. in central regions). We therefore aimed to determine the cellular location of a mTORC1-mediated phosphorylation event, RPS6Ser240/244, in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25g/kg protein, 0.75g/kg carbohydrate) alone (n=7;23±5yrs;76.8±3.6kg;13.6±3.8%BF, FED) or following a whole-body resistance exercise bout (n=7;22±2yrs;78.1±3.6kg;12.2±4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300min following anabolic stimuli. RPS6Ser240/244 phosphorylation measured by immunofluorescent staining or immunoblot was positively correlated (r=0.76, p<0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120min (~54% and ~138% respectively, p<0.05) but was greater in EXFED at both post-stimuli time points (p<0.05). The peripheral-central ratio of p-RPS6240/244 staining displayed a similar pattern, even when corrected for total RPS6 distribution, suggesting RPS6 phosphorylation occurs to a greater extent in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120min irrespective of stimulus (p=0.006) before returning to PRE at 300min. These data confirm that RPS6Ser240/244 phosphorylation occurs in the region of human muscle fibers to which mTOR translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 regulation in vivo.

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