Accelerated de novo sarcomere assembly by electric pulse stimulation in C2C12 myotubes

Exercise has beneficial effects on human health and is affected by two different pathways; motoneuron and endocrine. For the advancement of exercise research, in vitro exercise models are essential. We established two in vitro exercise models using C2C12 myotubes; EPS (electrical pulse stimulation) for a motoneuron model and clenbuterol, a specific β2 adrenergic receptor agonist, treatment for an endocrine model. For clenbuterol treatment, we found that Ppargc1a was induced only in low glucose media (1 mg/mL) using a 1-h treatment of 30 ng/mL clenbuterol. Global transcriptional changes of clenbuterol treatment were analyzed by RNA-seq and gene ontology analyses and indicated that mitogenesis and the PI3K-Akt pathway were enhanced, which is consistent with the effects of exercise. Cxcl1 and Cxcl5 were identified as candidate myokines induced by adrenaline. As for the EPS model, we compared 1 Hz of 1-pulse EPS and 1 Hz of 10-pulse EPS for 24 h and determined Myh gene expressions. Ten-pulse EPS induced higher Myh2 and Myh7 expression. Global transcriptional changes of 10-pulse EPS were also analyzed using RNA-seq, and gene ontology analyses indicated that CaMK signaling and hypertrophy pathways were enhanced, which is also consistent with the effects of exercise. In this paper, we provided two transcriptome results of in vitro exercise models and these databases will contribute to advances in exercise research.

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Effect of differentiation, de novo innervation, and electrical pulse stimulation on mRNA and protein expression of Na+,K+-ATPase, FXYD1, and FXYD5 in cultured human skeletal muscle cells

PLoS ONE ◽

10.1371/journal.pone.0247377 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0247377

Author(s):

Vid Jan ◽

Katarina Miš ◽

Natasa Nikolic ◽

Klemen Dolinar ◽

Metka Petrič ◽

...

Keyword(s):

Skeletal Muscle ◽

Mrna Expression ◽

Human Skeletal Muscle ◽

De Novo ◽

Muscle Cells ◽

Electrical Pulse ◽

Electrical Pulse Stimulation ◽

Human Skeletal Muscle Cells ◽

Pulse Stimulation ◽

Low Serum

Denervation reduces the abundance of Na+,K+-ATPase (NKA) in skeletal muscle, while reinnervation increases it. Primary human skeletal muscle cells, the most widely used model to study human skeletal muscle in vitro, are usually cultured as myoblasts or myotubes without neurons and typically do not contract spontaneously, which might affect their ability to express and regulate NKA. We determined how differentiation, de novo innervation, and electrical pulse stimulation affect expression of NKA (α and β) subunits and NKA regulators FXYD1 (phospholemman) and FXYD5 (dysadherin). Differentiation of myoblasts into myotubes under low serum conditions increased expression of myogenic markers CD56 (NCAM1), desmin, myosin heavy chains, dihydropyridine receptor subunit α1S, and SERCA2 as well as NKAα2 and FXYD1, while it decreased expression of FXYD5 mRNA. Myotubes, which were innervated de novo by motor neurons in co-culture with the embryonic rat spinal cord explants, started to contract spontaneously within 7–10 days. A short-term co-culture (10–11 days) promoted mRNA expression of myokines, such as IL-6, IL-7, IL-8, and IL-15, but did not affect mRNA expression of NKA, FXYDs, or myokines, such as musclin, cathepsin B, meteorin-like protein, or SPARC. A long-term co-culture (21 days) increased the protein abundance of NKAα1, NKAα2, FXYD1, and phospho-FXYD1Ser68 without attendant changes in mRNA levels. Suppression of neuromuscular transmission with α-bungarotoxin or tubocurarine for 24 h did not alter NKA or FXYD mRNA expression. Electrical pulse stimulation (48 h) of non-innervated myotubes promoted mRNA expression of NKAβ2, NKAβ3, FXYD1, and FXYD5. In conclusion, low serum concentration promotes NKAα2 and FXYD1 expression, while de novo innervation is not essential for upregulation of NKAα2 and FXYD1 mRNA in cultured myotubes. Finally, although innervation and EPS both stimulate contractions of myotubes, they exert distinct effects on the expression of NKA and FXYDs.

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LOW-INTENSITY EXERCISE ENHANCES MUSCULAR ANDROGEN/ANDROGEN RECEPTOR TO INHIBIT MYOSTATIN PATHWAY

Innovation in Aging ◽

10.1093/geroni/igz038.329 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

pp. S85-S86

Author(s):

Bo-Kyung Son ◽

Masato Eto ◽

Miya Oura ◽

Masahiro Akishita

Keyword(s):

Skeletal Muscle ◽

Androgen Receptor ◽

Electric Pulse ◽

Negative Regulator ◽

Muscle Size ◽

C2c12 Myotubes ◽

Protein Levels ◽

Low Intensity ◽

Low Intensity Exercise ◽

Response To Exercise

Abstract Background: Physical exercise is well documented to induce muscle size, strength, and energy metabolism. Although the contribution of systemic or local androgen in exercise-adapted muscle hypertrophy has been suggested, less is known about the molecular pathway of androgen in response to exercise. In the present study, we examined roles of androgen/androgen receptor (AR) after exercise, especially for the suppression of myostatin, a potent negative regulator of muscle mass. Methods and Results: To examine the effects of exercise, we employed low-intensity exercise in mice and electric pulse stimulation (EPS) in C2C12 myotubes. Both mRNA and protein levels of AR significantly increased in skeletal muscle of low-intensity exercised mice and C2C12 myotubes exposed to EPS. Production of testosterone and DHT from EPS-treated C2C12 myotubes was markedly increased. Of interest, we found that myostatin was clearly inhibited by EPS, and its inhibition was significantly abrogated by flutamide, a specific antagonist of AR. Furthermore, IL-6 and phospho-STAT3 (pSTAT3) expression, the downstream pathway of myostatin, were decreased by EPS and this was also reversed by flutamide. Similar downregulation of myostatin and IL-6 was seen in skeletal muscle of low-intensity exercised mice. Conclusion: Muscle AR expression and androgen production were increased by exercise and EPS treatment. As a mechanistical insight, it is suggested that AR inhibited myostatin expression transcriptionally, which downregulates IL-6/pSTAT3 pathway and thus contributes to the prevention of muscle degradation.

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Platelet activation using electric pulse stimulation

Journal of Trauma and Acute Care Surgery ◽

10.1097/ta.0000000000000322 ◽

2014 ◽

Vol 77 ◽

pp. S94-S100 ◽

Cited By ~ 16

Author(s):

Andrew S. Torres ◽

Antonio Caiafa ◽

Allen L. Garner ◽

Steve Klopman ◽

Nicole LaPlante ◽

...

Keyword(s):

Platelet Activation ◽

Electric Pulse ◽

Pulse Stimulation

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Muscle specific stress fibers give rise to sarcomeres and are mechanistically distinct from stress fibers in non-muscle cells

10.1101/235424 ◽

2017 ◽

Cited By ~ 1

Author(s):

Aidan M. Feinx ◽

Nilay Taneja ◽

Abigail C. Neininger ◽

Mike R. Visetsouk ◽

Benjamin R. Nixon ◽

...

Keyword(s):

Actin Polymerization ◽

De Novo ◽

Muscle Cells ◽

Stress Fiber ◽

Stress Fibers ◽

Myosin Filament ◽

Human Cardiomyocytes ◽

Fiber Assembly ◽

Filament Assembly ◽

Sarcomere Assembly

AbstractThe sarcomere is the basic contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating thin (i.e., actin) and thick (i.e., myosin) filament assembly during sarcomere formation. Thus, we developed an assay using human cardiomyocytes to test de novo sarcomere assembly. Using this assay, we report a population of muscle-specific stress fibers are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from these muscle stress fibers. This process requires formin-mediated but not Arp2/3-mediated actin polymerization and nonmuscle myosin IIB but not non-muscle myosin IIA. Furthermore, we show a short species of β cardiac myosin II filaments grows to form ~1.5 long filaments that then “stitch” together to form the stack of filaments at the core of the sarcomere (i.e., A-band). Interestingly, these are different from mechanisms that have previously been reported during stress fiber assembly in non-muscle cells. Thus, we provide a new model of cardiac sarcomere assembly based on distinct mechanisms of stress fiber regulation between non-muscle and muscle cells.

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