Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation

2006 ◽  
Vol 291 (1) ◽  
pp. C11-C17 ◽  
Author(s):  
Yen-Chih Huang ◽  
Robert G. Dennis ◽  
Keith Baar
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Relaxation Times ◽  
Three Dimensional ◽  
Low Frequency ◽  
Force Production ◽  
Force Frequency ◽  
Protein Markers ◽  
Myogenic Cells

In vitro studies have used protein markers to distinguish between myogenic cells isolated from fast and slow skeletal muscles. The protein markers provide some support for the hypothesis that satellite cells from fast and slow muscles are different, but the data are equivocal. To test this hypothesis directly, three-dimensional skeletal muscle constructs were engineered from myogenic cells isolated from fast tibialis anterior (TA) and slow soleus (SOL) muscles of rats and functionality was tested. Time to peak twitch tension (TPT) and half relaxation time (RT1/2) were ∼30% slower in constructs from the SOL. The slower contraction and relaxation times for the SOL constructs resulted in left shift of the force-frequency curve compared with those from the TA. Western blot analysis showed a 60% greater quantity of fast myosin heavy chain in the TA constructs. 14 days of chronic low-frequency electrical stimulation resulted in a 15% slower TPT and a 14% slower RT1/2, but no change in absolute force production in the TA constructs. In SOL constructs, slow electrical stimulation resulted in an 80% increase in absolute force production with no change in TPT or RT1/2. The addition of cyclosporine A did not prevent the increase in force in SOL constructs after chronic low-frequency electrical stimulation, suggesting that calcineurin is not responsible for the increase in force. We conclude that myogenic cells associated with a slow muscle are imprinted to produce muscle that contracts and relaxes slowly and that calcineurin activity cannot explain the response to a slow pattern of electrical stimulation.

Rapid formation of functional muscle in vitro using fibrin gels

2005 ◽  
Vol 98 (2) ◽  
pp. 706-713 ◽  
Author(s):  
Yen-Chih Huang ◽  
Robert G. Dennis ◽  
Lisa Larkin ◽  
Keith Baar
Keyword(s):  
Physiological Function ◽  
Three Dimensional ◽  
Force Production ◽  
Force Transducer ◽  
Force Frequency ◽  
Platinum Electrodes ◽  
Fibrin Gel ◽  
Novel Method

The transition of a muscle cell from a differentiated myotube into an adult myofiber is largely unstudied. This is primarily due to the difficulty of isolating specific developmental stimuli in vivo and the inability to maintain viable myotubes in culture for sufficient lengths of time. To address these limitations, a novel method for rapidly generating three-dimensional engineered muscles using fibrin gel casting has been developed. Myoblasts were seeded and differentiated on top of a fibrin gel. Cell-mediated contraction of the gel around artificial anchors placed 12 mm apart culminates 10 days after plating in a tubular structure of small myotubes (10-μm diameter) surrounded by a fibrin gel matrix. These tissues can be connected to a force transducer and electrically stimulated between parallel platinum electrodes to monitor physiological function. Three weeks after plating, the three-dimensional engineered muscle generated a maximum twitch force of 329 ± 26.3 μN and a maximal tetanic force of 805.8 ± 55 μN. The engineered muscles demonstrated normal physiological function including length-tension and force-frequency relationships. Treatment with IGF-I resulted in a 50% increase in force production, demonstrating that these muscles responded to hormonal interventions. Although the force production was maximal at 3 wk, constructs can be maintained in culture for up to 6 wk with no intervention. We conclude that fibrin-based gels provide a novel method to engineer three-dimensional functional muscle tissue and that these tissues may be used to model the development of skeletal muscle in vitro.

Calcium-independent phospholipase A2 modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells

2006 ◽  
Vol 100 (2) ◽  
pp. 399-405 ◽  
Author(s):  
Ming C. Gong ◽  
Sandrine Arbogast ◽  
Zhenheng Guo ◽  
Jeremy Mathenia ◽  
Wen Su ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mammalian Cells ◽  
Contractile Function ◽  
Muscle Fibers ◽  
Force Production ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Force Frequency ◽  
Striated Muscles

Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 μM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.

scholarly journals Recycled algae-based carbon materials as electroconductive 3D printed skeletal muscle tissue engineering scaffolds

2021 ◽  
Vol 32 (7) ◽  
Author(s):  
Selva Bilge ◽  
Emre Ergene ◽  
Ebru Talak ◽  
Seyda Gokyer ◽  
Yusuf Osman Donar ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Tissue Engineering ◽  
Electrical Stimulation ◽  
Muscle Tissue ◽  
Carbonaceous Material ◽  
Hydrothermal Carbonization ◽  
Skeletal Muscle Tissue ◽  
Myotube Formation ◽  
3D Printed

AbstractSkeletal muscle is an electrically and mechanically active tissue that contains highly oriented, densely packed myofibrils. The tissue has self-regeneration capacity upon injury, which is limited in the cases of volumetric muscle loss. Several regenerative therapies have been developed in order to enhance this capacity, as well as to structurally and mechanically support the defect site during regeneration. Among them, biomimetic approaches that recapitulate the native microenvironment of the tissue in terms of parallel-aligned structure and biophysical signals were shown to be effective. In this study, we have developed 3D printed aligned and electrically active scaffolds in which the electrical conductivity was provided by carbonaceous material (CM) derived from algae-based biomass. The synthesis of this conductive and functional CM consisted of eco-friendly synthesis procedure such as pre-carbonization and multi-walled carbon nanotube (MWCNT) catalysis. CM obtained from biomass via hydrothermal carbonization (CM-03) and its ash form (CM-03K) were doped within poly(ɛ-caprolactone) (PCL) matrix and 3D printed to form scaffolds with aligned fibers for structural biomimicry. Scaffolds were seeded with C2C12 mouse myoblasts and subjected to electrical stimulation during the in vitro culture. Enhanced myotube formation was observed in electroactive groups compared to their non-conductive counterparts and it was observed that myotube formation and myotube maturity were significantly increased for CM-03 group after electrical stimulation. The results have therefore showed that the CM obtained from macroalgae biomass is a promising novel source for the production of the electrically conductive scaffolds for skeletal muscle tissue engineering.

scholarly journals Bioengineered in vitro skeletal muscles as new tools for muscular dystrophies preclinical studies

2021 ◽  
Vol 12 ◽  
pp. 204173142098133
Author(s):  
Juan M. Fernández-Costa ◽  
Xiomara Fernández-Garibay ◽  
Ferran Velasco-Mallorquí ◽  
Javier Ramón-Azcón
Keyword(s):  
Skeletal Muscle ◽  
Tissue Engineering ◽  
Electrical Stimulation ◽  
Skeletal Muscles ◽  
Screening Tools ◽  
Muscular Dystrophies ◽  
Preclinical Studies ◽  
Technological Advances ◽  
Topographical Cues

Muscular dystrophies are a group of highly disabling disorders that share degenerative muscle weakness and wasting as common symptoms. To date, there is not an effective cure for these diseases. In the last years, bioengineered tissues have emerged as powerful tools for preclinical studies. In this review, we summarize the recent technological advances in skeletal muscle tissue engineering. We identify several ground-breaking techniques to fabricate in vitro bioartificial muscles. Accumulating evidence shows that scaffold-based tissue engineering provides topographical cues that enhance the viability and maturation of skeletal muscle. Functional bioartificial muscles have been developed using human myoblasts. These tissues accurately responded to electrical and biological stimulation. Moreover, advanced drug screening tools can be fabricated integrating these tissues in electrical stimulation platforms. However, more work introducing patient-derived cells and integrating these tissues in microdevices is needed to promote the clinical translation of bioengineered skeletal muscle as preclinical tools for muscular dystrophies.

scholarly journals Skeletal muscle-on-a-chip: an in vitro model to evaluate tissue formation and injury

Lab on a Chip ◽  
2017 ◽  
Vol 17 (20) ◽  
pp. 3447-3461 ◽  
Author(s):  
Gaurav Agrawal ◽  
Aereas Aung ◽  
Shyni Varghese
Keyword(s):  
Skeletal Muscle ◽  
Muscle Injury ◽  
In Vitro Model ◽  
Three Dimensional ◽  
Tissue Formation ◽  
Microfluidic Platform ◽  
Muscle Tissues ◽  
Vitro Model

We introduce a microfluidic platform in which we culture three-dimensional skeletal muscle tissues, while evaluating tissue formation and toxin-induced muscle injury.

scholarly journals Changes in Gene Expression of the MCU Complex Are Induced by Electrical Stimulation in Adult Skeletal Muscle

2021 ◽  
Vol 11 ◽  
Author(s):  
Esteban R. Quezada ◽  
Alexis Díaz-Vegas ◽  
Enrique Jaimovich ◽  
Mariana Casas
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Muscle Fibers ◽  
Low Frequency ◽  
Extracellular Atp ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Adult Skeletal Muscle ◽  
Muscle Plasticity

The slow calcium transient triggered by low-frequency electrical stimulation (ES) in adult muscle fibers and regulated by the extracellular ATP/IP3/IP3R pathway has been related to muscle plasticity. A regulation of muscular tropism associated with the MCU has also been described. However, the role of transient cytosolic calcium signals and signaling pathways related to muscle plasticity over the regulation of gene expression of the MCU complex (MCU, MICU1, MICU2, and EMRE) in adult skeletal muscle is completely unknown. In the present work, we show that 270 0.3-ms-long pulses at 20-Hz ES (and not at 90 Hz) transiently decreased the mRNA levels of the MCU complex in mice flexor digitorum brevis isolated muscle fibers. Importantly, when ATP released after 20-Hz ES is hydrolyzed by the enzyme apyrase, the repressor effect of 20 Hz on mRNA levels of the MCU complex is lost. Accordingly, the exposure of muscle fibers to 30 μM exogenous ATP produces the same effect as 20-Hz ES. Moreover, the use of apyrase in resting conditions (without ES) increased mRNA levels of MCU, pointing out the importance of extracellular ATP concentration over MCU mRNA levels. The use of xestospongin B (inhibitor of IP3 receptors) also prevented the decrease of mRNA levels of MCU, MICU1, MICU2, and EMRE mediated by a low-frequency ES. Our results show that the MCU complex can be regulated by electrical stimuli in a frequency-dependent manner. The changes observed in mRNA levels may be related to changes in the mitochondria, associated with the phenotypic transition from a fast- to a slow-type muscle, according to the described effect of this stimulation frequency on muscle phenotype. The decrease in mRNA levels of the MCU complex by exogenous ATP and the increase in MCU levels when basal ATP is reduced with the enzyme apyrase indicate that extracellular ATP may be a regulator of the MCU complex. Moreover, our results suggest that this regulation is part of the axes linking low-frequency stimulation with ATP/IP3/IP3R.

scholarly journals Interleukin 4 Moderately Affects Competence of Pluripotent Stem Cells for Myogenic Conversion

2019 ◽  
Vol 20 (16) ◽  
pp. 3932 ◽  
Author(s):  
Barbara Świerczek-Lasek ◽  
Jacek Neska ◽  
Agata Kominek ◽  
Łukasz Tolak ◽  
Tomasz Czajkowski ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Muscle Development ◽  
Embryonic Stem ◽  
Interleukin 4 ◽  
Myogenic Cells ◽  
The Impact

Pluripotent stem cells convert into skeletal muscle tissue during teratoma formation or chimeric animal development. Thus, they are characterized by naive myogenic potential. Numerous attempts have been made to develop protocols enabling efficient and safe conversion of pluripotent stem cells into functional myogenic cells in vitro. Despite significant progress in the field, generation of myogenic cells from pluripotent stem cells is still challenging—i.e., currently available methods require genetic modifications, animal-derived reagents, or are long lasting—and, therefore, should be further improved. In the current study, we investigated the influence of interleukin 4, a factor regulating inter alia migration and fusion of myogenic cells and necessary for proper skeletal muscle development and maintenance, on pluripotent stem cells. We assessed the impact of interleukin 4 on proliferation, selected gene expression, and ability to fuse in case of both undifferentiated and differentiating mouse embryonic stem cells. Our results revealed that interleukin 4 slightly improves fusion of pluripotent stem cells with myoblasts leading to the formation of hybrid myotubes. Moreover, it increases the level of early myogenic genes such as Mesogenin1, Pax3, and Pax7 in differentiating embryonic stem cells. Thus, interleukin 4 moderately enhances competence of mouse pluripotent stem cells for myogenic conversion.

Excitation-induced Ca2+uptake in rat skeletal muscle

1999 ◽  
Vol 276 (2) ◽  
pp. R331-R339 ◽  
Author(s):  
H. Gissel ◽  
T. Clausen
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Initial Rate ◽  
Low Frequency ◽  
Channel Blockers ◽  
Ca Uptake ◽  
Edl Muscle ◽  
Isotonic Contractions ◽  
Progressive Accumulation ◽  
Stimulation Of

In isolated rat extensor digitorum longus (EDL) muscle mounted for isometric contractions, chronic low-frequency electrical stimulation was found to lead to an increased uptake of45Ca (154% above control after 240 min) and a progressive accumulation of Ca2+ (85% above control after 240 min). In soleus, however, this treatment led to a small, but significant, increase in 45Ca uptake (30% above control after 180 min) but no significant accumulation of Ca2+. In muscles mounted for isotonic contractions without any external load, electrical stimulation gave rise to a larger45Ca uptake and accumulation of Ca2+ in both EDL and soleus. These uptakes of Ca2+ coincided with an accumulation of Na+. During isometric or isotonic contractions, stimulation at 40 Hz increased the initial (60 s) rate of 45Ca uptake in soleus muscle 15- and 30-fold, respectively. The stimulation-induced increase in 45Ca uptake was only reduced by 17% by the Ca2+-channel blockers nifedipine and verapamil but was blocked by tetrodotoxin. The initial rate of stimulation-induced 22Na and45Ca uptake was correlated ( r = 0.80; P < 0.003). Stimulation of Na+ channels with veratridine increased 45Ca uptake by 93 and 139% in soleus and EDL, respectively ( P < 0.001), effects that were abolished by tetrodotoxin. The results indicate that in skeletal muscle, excitation induces a considerable influx of Ca2+, mediated by Na+ channels.

Effects of pH on the length-dependent twitch potentiation in skeletal muscle

2002 ◽  
Vol 92 (3) ◽  
pp. 1293-1299 ◽  
Author(s):  
Dilson E. Rassier ◽  
Walter Herzog
Keyword(s):  
Skeletal Muscle ◽  
Force Production ◽  
Optimal Length ◽  
Twitch Potentiation ◽  
Length Dependence ◽  
Charge Potential ◽  
Before And After ◽  
Effects Of Ph ◽  
Isometric Twitch

When muscle is elongated, there is a length dependence of twitch potentiation and an increased Ca2+ sensitivity of the myofilaments. Changes in the charge potential of myofilaments, induced by a decrease in pH, are known to abolish the length dependence of Ca2+ sensitivity. This study was aimed at testing the hypothesis that a decrease in pH, and the concomitant loss of length dependence of Ca2+sensitivity, depresses the length dependence of staircase potentiation. In vitro, isometric twitch contractions of fiber bundles dissected from the mouse extensor digitorum longus, performed before and after 10 s of 10-Hz stimulation (i.e., the staircase potentiation protocol) were analyzed at five different lengths, ranging from optimal length for maximal force production ( L o; = 12 ± 0.7 mm) to L o + 1.2 mm ( L o + 10%). These measurements were made at an extracellular pH of 6.6, 7.4, and 7.8 (pH changes induced by altering the CO2 concentration of the bath solution). At pH 7.4 and 7.8, the degree of potentiation after 10-Hz stimulation showed a linear decrease with increased fiber bundle length ( r 2 = 0.95 and r 2 = 0.99, respectively). At pH 6.6, the length dependence of potentiation was abolished, and the slope of the length-potentiation relationship was not different from zero ( r 2 = 0.05). The results of this study indicate that length dependence of potentiation in intact skeletal muscle is abolished by lowering the pH. Because decreasing the pH decreases Ca2+ sensitivity and changes the charge potential of the filaments, the mechanism of length-dependent potentiation may be closely related to the length dependence of Ca2+sensitivity, and changes in the charge potential of the myofilaments may be important in regulating this relationship.

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