Electrical Pulse Stimulation of Primary Human Skeletal Muscle Cells

Nikolić, Nataša; Aas, Vigdis

doi:10.1007/978-1-4939-8897-6_2

Cited by 17 publications

(10 citation statements)

References 15 publications

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“…In EPS protocol 1, the myotubes were differentiated for 6 days, washed three times with PBS, and the media were changed to serum-free differentiation media before a single, bipolar pulses of 2 ms, with voltage 10 V and frequency 0.1 Hz were applied continuously for 24 h using a C-Dish™ carbon electrode connected to a C-Pace EP multi-channel culture pacer (IonOptix, Dublin, Ireland). In EPS protocol 2, the cells were differentiated for 5 days and exposed to EPS with 2 ms pulse duration, 30 V intensity, and frequency 1 Hz for the last 48 h of differentiation as previously described ( Nikolić et al, 2019 ). In both protocols, control myotubes cultured on 6-well CelBIND ® microplates were fitted with a C-Dish™ carbon electrode without connecting the C-Dish to the pulse generator.…”

Section: Methodsmentioning

confidence: 99%

Insight Into the Metabolic Adaptations of Electrically Pulse-Stimulated Human Myotubes Using Global Analysis of the Transcriptome and Proteome

Mengeste

Nikolić²,

Fernandez³

et al. 2022

Front. Physiol.

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Electrical pulse stimulation (EPS) has proven to be a useful tool to interrogate cell-specific responses to muscle contraction. In the present study, we aimed to uncover networks of signaling pathways and regulatory molecules responsible for the metabolic effects of exercise in human skeletal muscle cells exposed to chronic EPS. Differentiated myotubes from young male subjects were exposed to EPS protocol 1 (i.e. 2 ms, 10 V, and 0.1 Hz for 24 h), whereas myotubes from middle-aged women and men were exposed to protocol 2 (i.e. 2 ms, 30 V, and 1 Hz for 48 h). Fuel handling as well as the transcriptome, cellular proteome, and secreted proteins of EPS-treated myotubes from young male subjects were analyzed using a combination of high-throughput RNA sequencing, high-resolution liquid chromatography-tandem mass spectrometry, oxidation assay, and immunoblotting. The data showed that oxidative metabolism was enhanced in EPS-exposed myotubes from young male subjects. Moreover, a total of 81 differentially regulated proteins and 952 differentially expressed genes (DEGs) were observed in these cells after EPS protocol 1. We also found 61 overlapping genes while comparing the DEGs to mRNA expression in myotubes from the middle-aged group exposed to protocol 2, assessed by microarray. Gene ontology (GO) analysis indicated that significantly regulated proteins and genes were enriched in biological processes related to glycolytic pathways, positive regulation of fatty acid oxidation, and oxidative phosphorylation, as well as muscle contraction, autophagy/mitophagy, and oxidative stress. Additionally, proteomic identification of secreted proteins revealed extracellular levels of 137 proteins were changed in myotubes from young male subjects exposed to EPS protocol 1. Selected putative myokines were measured using ELISA or multiplex assay to validate the results. Collectively, our data provides new insight into the transcriptome, proteome and secreted proteins alterations following in vitro exercise and is a valuable resource for understanding the molecular mechanisms and regulatory molecules mediating the beneficial metabolic effects of exercise.

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Section: Methodsmentioning

confidence: 99%

Insight Into the Metabolic Adaptations of Electrically Pulse-Stimulated Human Myotubes Using Global Analysis of the Transcriptome and Proteome

Mengeste

Nikolić²,

Fernandez³

et al. 2022

Front. Physiol.

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“…“ In vitro exercise models”, using myotubes contracting in response to the application of electric pulse stimulation (EPS) 1 , have been widely utilized for investigating the impacts of actual contractile activity on muscle cell properties including exercise-inducible myokine secretion 2 , hypertrophy 3 , sarcomere lesions 4 and beneficial anti-lipotoxicity effects 5 . Although most prior studies used murine muscle cell lines such as mouse C2C12 cells 6–8 , several investigations have also been conducted using human muscle cells and provided compelling evidence that “ in vitro exercise models” are potentially applicable not only to murine muscle cells but also muscle cells obtained from human subjects 3,9–15 .…”

Section: Introductionmentioning

confidence: 99%

In vitro exercise model using contractile human and mouse hybrid myotubes

Chen

Nyasha

Koide

et al. 2019

Sci Rep

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Contraction of cultured myotubes with application of electric pulse stimulation (EPS) has been utilized for investigating cellular responses associated with actual contractile activity. However, cultured myotubes derived from human subjects often exhibit relatively poor EPS-evoked contractile activity, resulting in minimal contraction-inducible responses ( i . e . myokine secretion). We herein describe an “ in vitro exercise model”, using hybrid myotubes comprised of human myoblasts and murine C2C12 myoblasts, exhibiting vigorous contractile activity in response to EPS. Species-specific analyses including RT-PCR and the BioPlex assay allowed us to separately evaluate contraction-inducible gene expressions and myokine secretions from human and mouse constituents of hybrid myotubes. The hybrid myotubes, half of which had arisen from primary human satellite cells obtained from biopsy samples, exhibited remarkable increases in the secretions of human cytokines (myokines) including interleukins (IL-6, IL-8, IL-10, and IL16), CXC chemokines (CXCL1, CXCL2, CXCL5, CXCL6, CXCL10), CC chemokines (CCL1, CCL2, CCL7, CCL8, CCL11, CCL13, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL25, CCL27), and IFN-γ in response to EPS-evoked contractile activity. Together, these results indicate that inadequacies arising from human muscle cells are effectively overcome by fusing them with murine C2C12 cells, thereby supporting the development of contractility and the resulting cellular responses of human-origin muscle cells. Our approach, using hybrid myotubes, further expands the usefulness of the “ in vitro exercise model”.

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“…To directly analyse the movement of proteins following contraction, confocal microscopy has been employed within various skeletal muscle systems [55][56][57]. To detect a specific protein, a fluorescent tag is inserted into the coding region of the gene leading to the expression of a protein containing a conjugated tag that is detectable via absorption and emission of specific wavelengths of light [58].…”

Section: Electrical Stimulated Contraction and Microscopymentioning

confidence: 99%

“…Additionally, fluorescent tags can often bind together and aggregate leading to incorrect localisation and cellular toxicity [59]. The main drawback of this system is the inability of it to be utilised within in vivo muscle, with imaging only possible in ex vivo and in vitro skeletal muscle systems that contract through electrical stimulation [55,60]. While visible myotubule shortening occurs with electrical stimulation, it is debatable whether this process is equivalent to that of contraction within in vivo tissue, with several important biomarkers of mitochondrial biogenesis unchanged [61].…”

Section: Electrical Stimulated Contraction and Microscopymentioning

confidence: 99%

Transcription Factor Movement and Exercise-Induced Mitochondrial Biogenesis in Human Skeletal Muscle: Current Knowledge and Future Perspectives

Taylor

Bishop

2022

IJMS

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In response to exercise, the oxidative capacity of mitochondria within skeletal muscle increases through the coordinated expression of mitochondrial proteins in a process termed mitochondrial biogenesis. Controlling the expression of mitochondrial proteins are transcription factors—a group of proteins that regulate messenger RNA transcription from DNA in the nucleus and mitochondria. To fulfil other functions or to limit gene expression, transcription factors are often localised away from DNA to different subcellular compartments and undergo rapid movement or accumulation only when required. Although many transcription factors involved in exercise-induced mitochondrial biogenesis have been identified, numerous conflicting findings and gaps exist within our knowledge of their subcellular movement. This review aims to summarise and provide a critical analysis of the published literature regarding the exercise-induced movement of transcription factors involved in mitochondria biogenesis in skeletal muscle.

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Electrical Pulse Stimulation of Primary Human Skeletal Muscle Cells

Cited by 17 publications

References 15 publications

Insight Into the Metabolic Adaptations of Electrically Pulse-Stimulated Human Myotubes Using Global Analysis of the Transcriptome and Proteome

Insight Into the Metabolic Adaptations of Electrically Pulse-Stimulated Human Myotubes Using Global Analysis of the Transcriptome and Proteome

In vitro exercise model using contractile human and mouse hybrid myotubes

Transcription Factor Movement and Exercise-Induced Mitochondrial Biogenesis in Human Skeletal Muscle: Current Knowledge and Future Perspectives

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