Optimal Ultrasound Exposure Conditions for Maximizing C2C12 Muscle Cell Proliferation and Differentiation.

Described here is an in vitro systematic investigation of the effects on C2C12 myoblasts of exposure to finely controlled and repeatable low-intensity pulsed ultrasound of different frequencies (500 kHz, 1 MHz, 3 MHz and 5 MHz) and different intensities (250, 500 and 1000 mW/cm2). An in-house stimulation system and an ultrasound-transparent cell culture well minimized reflections and attenuations, allowing precise control of ultrasound delivery. Results indicated that a 3 MHz stimulation at 1 W/cm2 intensity maximized cell proliferation in comparison with the other exposure conditions and untreated controls. In contrast, cell differentiation and the consequent formation of multinucleated myotubes were maximized by 1 MHz stimulation at 500 mW/cm2 intensity. The highly controlled exposure conditions employed allowed precise correlation of the ultrasound delivery to the bio-effects produced, thus overcoming the inconsistency of some results available in the literature and contributing to the potential of ultrasound treatment for muscle therapy and regeneration.

Skeletal Muscle Cell Behavior After Physical Agent Treatments.

Apoptosis is essential for skeletal muscle development and homeostasis. It has been frequently involved in several muscle myopathies and sarcopenia, as well as in denervation, in disuse and acute strenuous or eccentric physical exercise. In this work skeletal muscle cell death, induced in vitro by a variety of physical triggers, has been investigated. C2C12 myoblasts and myotubes were exposed to UVB for 30 min, hyperthermia for 1 h at 43 °C, low pH for 3 h, hypothermia for 4h at 0 - 6°C, all followed by 2 - 4 h recovery. Their effects have been analysed by means of morpho- functional and molecular approaches. After UVB radiation, hyperthermia and acidosis, morphological apoptotic features and in situ DNA fragmentation appeared, more evident in myoblasts. Interestingly, apoptotic, non apoptotic and necrotic nuclei could be occasionally observed within the same myotube. Low pH induced apoptosis and necrosis, both characterized by swollen nuclei. In all these experimental conditions, the molecular investigations revealed a caspase pathway involvement in inducing cell death. Differently, hypothermia showed a scant and initial chromatin margination, in the presence of a diffused autophagic component. In this case, in situ DNA fragmentation and caspase activation have not been detected. Myoblasts and myotubes appeared sensitive to physical agents, some of which, induced apoptotic cell death. Moreover, hypothermia exposure seemed to enhance autophagic response, thus representing a way to delay trauma-correlated muscle inflammation. This study permits to highlight skeletal muscle cell behavior in response to physical agents, by adding important information to muscle cell death knowledge. UVB radiation and hyperthermia, usually used in clinical therapy, have also adverse effects on skeletal muscle such as myonuclei loss and cell death, contributing to muscle mass decrease. Acidosis occurs physiologically in muscular fatigue, reducing not only the athlete performance, but causing muscle cell damage or death too. Finally, hypothermia, stimulating the autophagic response, could have a key role in muscle injury prevention.

Simple micropatterning method for enhancing fusion efficiency and responsiveness to electrical stimulation of C2C12 myotubes.

Cultured myotubes induced in vitro from myoblast cell lines have been widely used to investigate muscle functional properties and disease-related biological phenotypes. Until now, several cell patterning techniques have been applied to regulate in vitro myotube structures. However, these previous studies required specific geometry patterns or soft materials for inducing efficient myotube formation. Thus, more simple and easy handling method will be promising. In this study, we aimed to provide a method to form C2C12 myotubes with regulated sizes and orientations in simple line patterns. We used a poly(dimethylsiloxane) (PDMS) stamp and a 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer solution to fabricate line patterns for myotube formation onto a culture dish. We confirmed that C2C12 myotubes of well-defined size and orientation were reproducibly formed. In particular, myotubes formed in the micropatterned lines showed the increased fusion efficiency. Then, functional dynamics in the micropatterned myotubes were detected and analyzed using a calcium imaging method. We confirmed micropatterning in line patterns enhanced the responsiveness of myotubes to external electrical stimulations. These results indicate that micropatterning myoblasts with the MPC polymer is a simple and effective method to form functional myotube networks.

Induction of skeletal muscle differentiation in vitro by therapeutic ultrasound.

Therapeutic ultrasound (TU) has been used for the last 50 y in rehabilitation, including treatment of soft tissues. Ultrasound waves can be employed in two different modes of operation, continuous and pulsed, which produce both thermal and non-thermal effects. Despite the large-scale use of TU, there are few scientific studies on its biologic effects during skeletal muscle differentiation. To better analyze the cellular effects of TU, we decided to follow cells in vitro. The main purpose of this study was to evaluate the effects of TU in primary chick myogenic cell cultures using phase contrast optical microscopy and immunofluorescence microscopy, followed by image analysis and quantification. Our results indicate that TU can stimulate the differentiation of skeletal muscle cells in vitro, as measured by the thickness of multinucleated myotubes, the ratio of mononucleated cells to multinucleated cells and expression of the muscle-specific protein desmin. This study is a first step toward a metrologic and science-based protocol for cell treatment under different ultrasound field exposures.

ELF-MF transiently increases skeletal myoblast migration: possible role of calpain system.

PURPOSE: Cell migration is crucial for myogenesis since it is required for the alignment and fusion of myoblast. Ca(2+) signals are involved in regulating myoblast migration and an extremely low frequency (ELF) magnetic field (MF) increases intracellular calcium levels in C2C12 myoblast. This study was aimed at investigating whether ELF-MF could affect myoblast migration. As calpains contribute to the regulation of myoblast motility, the effect of ELF-MF on µ- and m-calpain was also investigated. MATERIALS AND METHODS: The effect of ELF-MF (1 mT; 50 Hz) on C2C12 cell motility was observed by wound-healing assay. Protein expression of calpains, calpastatin, myristoylated alanine-rich C-kinase substrate (MARCKS) and vinculin were examined by Western blot analysis. Casein zymography and immunofluorescence analysis were carried out to evaluate, respectively, activity levels of calpains and intracellular distribution of calpains, calpastatin and actin. RESULTS: Exposure to ELF-MF resulted in a transient but significant increase of myoblast migration. This stimulatory effect was associated with a marked increase of µ- and m-calpain activity followed by the concomitant variation in their subcellular localization. No significant changes in intracellular distribution and protein levels of calpastatin were detected. Finally, a significant decrease of MARCKS expression and modifications of actin dynamics were reported. CONCLUSIONS: This study clearly outlines an involvement of calpains in ELF-MF-mediated myoblast migration.

Formation and optogenetic control of engineered 3D skeletal muscle bioactuators.

Densely arrayed skeletal myotubes are activated individually and as a group using precise optical stimulation with high spatiotemporal resolution. Skeletal muscle myoblasts are genetically encoded to express a light-activated cation channel, Channelrhodopsin-2, which allows for spatiotemporal coordination of a multitude of skeletal myotubes that contract in response to pulsed blue light. Furthermore, ensembles of mature, functional 3D muscle microtissues have been formed from the optogenetically encoded myoblasts using a high-throughput device. The device, called "skeletal muscle on a chip", not only provides the myoblasts with controlled stress and constraints necessary for muscle alignment, fusion and maturation, but also facilitates the measurement of forces and characterization of the muscle tissue. We measured the specific static and dynamic stresses generated by the microtissues and characterized the morphology and alignment of the myotubes within the constructs. The device allows testing of the effect of a wide range of parameters (cell source, matrix composition, microtissue geometry, auxotonic load, growth factors and exercise) on the maturation, structure and function of the engineered muscle tissues in a combinatorial manner. Our studies integrate tools from optogenetics and microelectromechanical systems (MEMS) technology with skeletal muscle tissue engineering to open up opportunities to generate soft robots actuated by a multitude of spatiotemporally coordinated 3D skeletal muscle microtissues.

No effect of low-level lasers on in vitro myoblast culture.

Effects of phototherapy using low-level lasers depend on irradiation parameters and the type of laser used. The aim of the present study was to evaluate the effect of phototherapy on the proliferation of cultured C2C12 myoblasts under different nutritional conditions using low-level GaAlAs and InGaAlP lasers with different parameters and incubation periods. C2C12 cells cultured in regular and nutrient-deficient medium were irradiated with low-level GaAlAs (780 nm) and InGaA1P (660 nm) lasers with energy densities of 3.8, 6.3 and 10 J/cm2, and 3.8, 10 and 17.5 J/cm2, respectively. Cell proliferation was assessed 48 and 72 h after irradiation by MTT assay. There were no significant differences in cell proliferation between laser-treated myoblasts and control cultures for any of the parameters and incubation periods. Further studies are necessary to determine the correct laser parameters for optimizing the biostirhulation of myoblasts.

Use of pifithrin to inhibit p53-mediated signalling of TNF in dystrophic muscles of mdx mice.

Tumour Necrosis Factor (TNF) plays a major role in exacerbating necrosis of dystrophic muscle; however, the precise molecular mechanism underlying this effect of TNF is unknown. This study investigates the role that p53 plays in TNF-mediated necrosis of dystrophic myofibres by inhibiting p53 using pifithrin-alpha and three pifithrin-beta analogues. Tissue culture studies using C2C12 myoblasts established that pifithrin-alpha was toxic to differentiating myoblasts at concentrations greater than 10 muM. While non-toxic concentrations of pifithrin-alpha did not prevent the TNF-mediated inhibition of myoblast differentiation, Western blots indicated that nuclear levels of p53 were higher in TNF-treated myoblasts indicating that TNF does elevate p53. In contrast, in vivo studies in adult mdx mice showed that pifithrin-alpha significantly reduced myofibre necrosis that resulted from voluntary wheel running over 48 h. These results support the hypothesis that p53 plays some role in TNF-mediated necrosis of dystrophic muscle and present a potential new target for therapeutic interventions.

Low-level laser irradiation promotes the recovery of atrophied gastrocnemius skeletal muscle in rats.

Low-level laser (LLL) irradiation promotes proliferation of muscle satellite cells, angiogenesis and expression of growth factors. Satellite cells, angiogenesis and growth factors play important roles in the regeneration of muscle. The objective of this study was to examine the effect of LLL irradiation on rat gastrocnemius muscle recovering from disuse muscle atrophy. Eight-week-old rats were subjected to hindlimb suspension for 2 weeks, after which they were released and recovered. During the recovery period, rats underwent daily LLL irradiation (Ga-Al-As laser; 830 nm; 60 mW; total, 180 s) to the right gastrocnemius muscle through the skin. The untreated left gastrocnemius muscle served as the control. In conjunction with LLL irradiation, 5-bromo-2-deoxyuridine (BrdU) was injected subcutaneously to label the nuclei of proliferating cells. After 2 weeks, myofibre diameters of irradiated muscle increased in comparison with those of untreated muscle, but did not recover back to normal levels. Additionally, in the superficial region of the irradiated muscle, the number of capillaries and fibroblast growth factor levels exhibited significant elevation relative to those of untreated muscle. In the deep region of irradiated muscle, BrdU-positive nuclei of satellite cells and/or myofibres increased significantly relative to those of the untreated muscle. The results of this study suggest that LLL irradiation can promote recovery from disuse muscle atrophy in association with proliferation of satellite cells and angiogenesis.

Effect of tumor necrosis factor alpha on electrically induced calcium transients elicited in C2C12 skeletal myotubes.

Diseases involving chronic inflammation can lead to prolonged exposure of skeletal muscle to inflammatory cytokines such as tumor necrosis factor alpha (TNFalpha), which may contribute to the skeletal muscle weakness seen in these conditions. In this study we examined the effect of a prolonged exposure to TNFalpha on intracellular Ca(2+) transients elicited in skeletal C(2)C(12) myotubes. A 48-h exposure to TNFalpha (10 ng/mL) significantly reduced the peaks, time to peak, and rate of Ca(2+) decay of electrically induced Ca(2+) transients elicited in C(2)C(12) skeletal myotubes. TNFalpha exposure had no significant effect on the resting Ca(2+) levels. The results of this study indicate that prolonged exposure to TNFalpha decreases sarcoplasmic reticulum Ca(2+) release in cultured skeletal muscle cells. This altered Ca(2+) release could contribute to the muscle weakness found in conditions involving chronic inflammation.