Matrigel 3D bioprinting of contractile human skeletal muscle models recapitulating exercise and pharmacological responses

AbstractA key to enhance the low translatability of preclinical drug discovery are in vitro human three-dimensional (3D) microphysiological systems (MPS). Here, we show a new method for automated engineering of 3D human skeletal muscle models in microplates and functional compound screening to address the lack of muscle wasting disease medication. To this end, we adapted our recently described 24-well plate 3D bioprinting platform with a printhead cooling system to allow microvalve-based drop-on-demand printing of cell-laden Matrigel containing primary human muscle precursor cells. Mini skeletal muscle models develop within a week exhibiting contractile, striated myofibers aligned between two attachment posts. As an in vitro exercise model, repeated high impact stimulation of contractions for 3 h by a custom-made electrical pulse stimulation (EPS) system for 24-well plates induced interleukin-6 myokine expression and Akt hypertrophy pathway activation. Furthermore, the known muscle stimulators caffeine and Tirasemtiv acutely increase EPS-induced contractile force of the models. This validated new human muscle MPS will benefit development of drugs against muscle wasting diseases. Moreover, our Matrigel 3D bioprinting platform will allow engineering of non-self-organizing complex human 3D MPS.

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Localization and function of ATP-sensitive potassium channels in human skeletal muscle

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00303.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. R558-R563 ◽

Cited By ~ 41

Author(s):

Jens Jung Nielsen ◽

Michael Kristensen ◽

Ylva Hellsten ◽

Jens Bangsbo ◽

Carsten Juel

Keyword(s):

Skeletal Muscle ◽

Muscle Activity ◽

Human Skeletal Muscle ◽

Knee Extensor ◽

Human Muscle ◽

Functional Importance ◽

Gradient Technique ◽

And Function

The present study investigated the localization of ATP-sensitive K+ (KATP) channels in human skeletal muscle and the functional importance of these channels for human muscle K+ distribution at rest and during muscle activity. Membrane fractionation based on the giant vesicle technique or the sucrose-gradient technique in combination with Western blotting demonstrated that the KATP channels are mainly located in the sarcolemma. This localization was confirmed by immunohistochemical measurements. With the microdialysis technique, it was demonstrated that local application of the KATP channel inhibitor glibenclamide reduced ( P < 0.05) interstitial K+ at rest from ∼4.5 to 4.0 mM, whereas the concentration in the control leg remained constant. Glibenclamide had no effect on the interstitial K+ accumulation during knee-extensor exercise at a power output of 60 W. In contrast to in vitro conditions, the present study demonstrated that under in vivo conditions the KATP channels are active at rest and contribute to the accumulation of interstitial K+.

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Myostatin promotes the wasting of human myoblast cultures through promoting ubiquitin-proteasome pathway-mediated loss of sarcomeric proteins

AJP Cell Physiology ◽

10.1152/ajpcell.00114.2011 ◽

2011 ◽

Vol 301 (6) ◽

pp. C1316-C1324 ◽

Cited By ~ 61

Author(s):

Sudarsanareddy Lokireddy ◽

Vincent Mouly ◽

Gillian Butler-Browne ◽

Peter D. Gluckman ◽

Mridula Sharma ◽

...

Keyword(s):

Skeletal Muscle ◽

Human Skeletal Muscle ◽

Muscle Wasting ◽

Negative Regulator ◽

Potent Inducer ◽

Ubiquitin Proteasome Pathway ◽

Skeletal Muscle Wasting ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

Myostatin is a negative regulator of skeletal muscle growth and in fact acts as a potent inducer of “cachectic-like” muscle wasting in mice. The mechanism of action of myostatin in promoting muscle wasting has been predominantly studied in murine models. Despite numerous reports linking elevated levels of myostatin to human skeletal muscle wasting conditions, little is currently known about the signaling mechanism(s) through which myostatin promotes human skeletal muscle wasting. Therefore, in this present study we describe in further detail the mechanisms behind myostatin regulation of human skeletal muscle wasting using an in vitro human primary myotube atrophy model. Treatment of human myotube populations with myostatin promoted dramatic myotubular atrophy. Mechanistically, myostatin-induced myotube atrophy resulted in reduced p-AKT concomitant with the accumulation of active dephosphorylated Forkhead Box-O (FOXO1) and FOXO3. We further show that addition of myostatin results in enhanced activation of atrogin-1 and muscle-specific RING finger protein 1 (MURF1) and reduced expression of both myosin light chain (MYL) and myosin heavy chain (MYH). In addition, we found that myostatin-induced loss of MYL and MYH proteins is dependent on the activity of the proteasome and mediated via SMAD3-dependent regulation of FOXO1 and atrogin-1. Therefore, these data suggest that the mechanism through which myostatin promotes muscle wasting is very well conserved between species, and that myostatin-induced human myotube atrophy is mediated through inhibition of insulin-like growth factor (IGF)/phosphoinositide 3-kinase (PI3-K)/AKT signaling and enhanced activation of the ubiquitin-proteasome pathway and elevated protein degradation.

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Recent Trends in Biofabrication Technologies for Studying Skeletal Muscle Tissue-Related Diseases

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.782333 ◽

2021 ◽

Vol 9 ◽

Author(s):

Seungyeun Cho ◽

Jinah Jang

Keyword(s):

Skeletal Muscle ◽

Muscle Tissue ◽

Motor Neurons ◽

Close Contact ◽

Human Muscle ◽

Skeletal Muscle Tissue ◽

Mechanism Study ◽

Muscle Diseases ◽

Muscle Models

In native skeletal muscle, densely packed myofibers exist in close contact with surrounding motor neurons and blood vessels, which are embedded in the fibrous connective tissue. In comparison to conventional two-dimensional (2D) cultures, the three-dimensional (3D) engineered skeletal muscle models allow structural and mechanical resemblance with native skeletal muscle tissue by providing geometric confinement and physiological matrix stiffness to the cells. In addition, various external stimuli applied to these models enhance muscle maturation along with cell–cell and cell–extracellular matrix interaction. Therefore, 3D in vitro muscle models can adequately recapitulate the pathophysiologic events occurring in tissue–tissue interfaces inside the native skeletal muscle such as neuromuscular junction. Moreover, 3D muscle models can induce pathological phenotype of human muscle dystrophies such as Duchenne muscular dystrophy by incorporating patient-derived induced pluripotent stem cells and human primary cells. In this review, we discuss the current biofabrication technologies for modeling various skeletal muscle tissue-related diseases (i.e., muscle diseases) including muscular dystrophies and inflammatory muscle diseases. In particular, these approaches would enable the discovery of novel phenotypic markers and the mechanism study of human muscle diseases with genetic mutations.

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Comparative analysis of different hydrogels for the bioprinting of 3D in vitro skeletal muscle models

Biomedical Science and Engineering ◽

10.4081/bse.2021.146 ◽

2021 ◽

Vol 4 (s1) ◽

Author(s):

Flaminia Aliberti ◽

Lorenza Rinvenuto ◽

Giada Loi ◽

Laura Benedetti ◽

Franca Scocozza ◽

...

Keyword(s):

Skeletal Muscle ◽

Comparative Analysis ◽

Muscle Cells ◽

3D Bioprinting ◽

Proliferation And Differentiation ◽

3D Em ◽

Muscle Models

In this study we demonstrated an application of 3D Bioprinting using different commercially available hydrogels (CELLINK AB, Sweden) with the aim to identify the most suitable biomaterial for the proliferation and differentiation of murine muscle cells (C2C12).

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Engineering a 3D in vitro model of human skeletal muscle at the single fiber scale

PLoS ONE ◽

10.1371/journal.pone.0232081 ◽

2020 ◽

Vol 15 (5) ◽

pp. e0232081 ◽

Cited By ~ 2

Author(s):

Anna Urciuolo ◽

Elena Serena ◽

Rusha Ghua ◽

Susi Zatti ◽

Monica Giomo ◽

...

Keyword(s):

Skeletal Muscle ◽

Human Skeletal Muscle ◽

In Vitro Model ◽

Single Fiber ◽

3D In Vitro Model ◽

Vitro Model

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An in vitro human skeletal muscle model: coculture of myotubes,neuron-like cells, and the capillary network

TURKISH JOURNAL OF BIOLOGY ◽

10.3906/biy-1611-22 ◽

2017 ◽

Vol 41 ◽

pp. 514-525

Author(s):

Ayşe Burcu ERTAN ◽

Halime KENAR ◽

Tahsin BEYZADEOĞLU ◽

Fatma Neşe KÖK ◽

Gamze TORUN KÖSE

Keyword(s):

Skeletal Muscle ◽

Human Skeletal Muscle ◽

Capillary Network ◽

Muscle Model

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FGF-2–dependent signaling activated in aged human skeletal muscle promotes intramuscular adipogenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2021013118 ◽

2021 ◽

Vol 118 (37) ◽

pp. e2021013118 ◽

Cited By ~ 1

Author(s):

Sebastian Mathes ◽

Alexandra Fahrner ◽

Umesh Ghoshdastider ◽

Hannes A. Rüdiger ◽

Michael Leunig ◽

...

Keyword(s):

Skeletal Muscle ◽

Transcriptional Activation ◽

Human Skeletal Muscle ◽

Muscle Growth ◽

Muscle Cells ◽

Adeno Associated Virus ◽

Adult Skeletal Muscle

Aged skeletal muscle is markedly affected by fatty muscle infiltration, and strategies to reduce the occurrence of intramuscular adipocytes are urgently needed. Here, we show that fibroblast growth factor-2 (FGF-2) not only stimulates muscle growth but also promotes intramuscular adipogenesis. Using multiple screening assays upstream and downstream of microRNA (miR)-29a signaling, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. Genetic deletions in muscle cells and adeno-associated virus–mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of fibro/adipogenic progenitors in vitro and intramuscular adipose tissue (IMAT) formation in vivo. Skeletal muscle from human donors aged >75 y versus <55 y showed activation of FGF-2–dependent signaling and increased IMAT. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a pathway to combat fat accumulation in aged human skeletal muscle.

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Ovarian cancer ascites induces skeletal muscle wasting in vitro and reflects sarcopenia in patients

Journal of Cachexia Sarcopenia and Muscle ◽

10.1002/jcsm.12885 ◽

2021 ◽

Author(s):

Jorne Ubachs ◽

Wouter R.P.H. Worp ◽

Rianne D.W. Vaes ◽

Kenneth Pasmans ◽

Ramon C. Langen ◽

...

Keyword(s):

Ovarian Cancer ◽

Skeletal Muscle ◽

Muscle Wasting ◽

Skeletal Muscle Wasting

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Microdialysis of skeletal muscle at rest

Proceedings of The Nutrition Society ◽

10.1017/s0029665199001226 ◽

1999 ◽

Vol 58 (4) ◽

pp. 919-923 ◽

Cited By ~ 28

Author(s):

Jan Henriksson

Keyword(s):

Skeletal Muscle ◽

Blood Flow ◽

Human Skeletal Muscle ◽

Human Metabolism ◽

High Expectations ◽

Perfusate Flow ◽

Muscle Research ◽

Interstitial Glucose

Techniques in human skeletal muscle research are by necessity predominantly 'descriptive'.Microdialysis has raised high expectations that it could meet the demand for a method that allows 'mechanistic' investigations to be performed in human skeletal muscle. In the present review, some views are given on how well the initial expectations on the use of the microdialysis technique in skeletal muscle have been fulfilled, and the areas in which additional work is needed in order to validate microdialysis as an important metabolic technique in this tissue. The microdialysis catheter has been equated to an artificial blood vessel, which is introduced into the tissue. By means of this 'vessel' the concentrations of compounds in the interstitial space can be monitored. The concentration of substances in the collected samples is dependent on the rate of perfusate flow. When perfusate flow is slow enough to allow complete equilibration between interstitial and perfusate fluids, the concentration in the perfusate is maximal and identical to the interstitial concentration. Microdialysis data may be influenced by changes in blood flow, especially in instances where the tissue diffusivity limits the recovery in vivo, i.e. when recovery in vitro is 100 %, whereas the recovery in vivo is less than 100 %. Microdialysis data indicate that a significant arterial-interstitial glucose concentration gradient exists in skeletal muscle but not in adipose tissue at rest. While the concentrations of glucose and lactate in the dialysate from skeletal muscle are close to the expected values, the glycerol values obtained for muscle are still puzzling. Ethanol added to the perfusate will be cleared by the tissue at a rate that is determined by the nutritive blood flow (the microdialysis ethanol technique). It is concluded that microdialysis of skeletal muscle has become an important technique for mechanistic studies in human metabolism and nutrition.

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