Effects of different protocols of defocused high-power laser on the viability and migration of myoblasts-a comparative in vitro study.

The aim of the present study was to analyze for the first time the effect of photobiomodulation therapy (PBMT) using defocused high-power laser (DHPL) in myoblast cell line C2C12 viability and migration and compare them with low-power laser therapy. Cells were divided into 9 groups: Sham irradiation 10% fetal bovine serum (FBS); Sham irradiation 5%FBS; low-power laser 0.1 W; DHPL 810 1 W; DHPL 810 2 W; DHPL 980 1 W; DHPL 980 2 W; DHPL dual 1 W; DHPL dual 2 W. To simulate stress conditions, all groups exposed to irradiation were maintained in DMEM 5% FBS. The impact of therapies on cell viability was assessed through sulforhodamine B assay and on cells migration through scratch assays and time-lapse. Myoblast viability was not modified by PBMT protocols. All PBMT protocols were able to accelerate the scratch closure after 6 and 18 h of the first irradiation (p < 0.001). Also, an increase in migration speed, with a more pronounced effect of DHPL laser using dual-wavelength protocol with 2 W was observed (p < 0.001). In conclusion, the diverse PBMT protocols used in this study accelerated the C2C12 myoblasts migration, with 2-W dual-wavelength outstanding as the most effective protocol tested. Benefits from treating muscle injuries with PBMT appear to be related to its capacity to induce cell migration without notable impact on cell viability.

Molecular and Metabolic Mechanism of Low-Intensity Pulsed Ultrasound Improving Muscle Atrophy in Hindlimb Unloading Rats.

Low-intensity pulsed ultrasound (LIPUS) has been proved to promote the proliferation of myoblast C2C12. However, whether LIPUS can effectively prevent muscle atrophy has not been clarified, and if so, what is the possible mechanism. The aim of this study is to evaluate the effects of LIPUS on muscle atrophy in hindlimb unloading rats, and explore the mechanisms. The rats were randomly divided into four groups: normal control group (NC), hindlimb unloading group (UL), hindlimb unloading plus 30 mW/cm2 LIPUS irradiation group (UL + 30 mW/cm2), hindlimb unloading plus 80 mW/cm2 LIPUS irradiation group (UL + 80 mW/cm2). The tails of rats in hindlimb unloading group were suspended for 28 days. The rats in the LIPUS treated group were simultaneously irradiated with LIPUS on gastrocnemius muscle in both lower legs at the sound intensity of 30 mW/cm2 or 80 mW/cm2 for 20 min/d for 28 days. C2C12 cells were exposed to LIPUS at 30 or 80 mW/cm2 for 5 days. The results showed that LIPUS significantly promoted the proliferation and differentiation of myoblast C2C12, and prevented the decrease of cross-sectional area of muscle fiber and gastrocnemius mass in hindlimb unloading rats. LIPUS also significantly down regulated the expression of MSTN and its receptors ActRIIB, and up-regulated the expression of Akt and mTOR in gastrocnemius muscle of hindlimb unloading rats. In addition, three metabolic pathways (phenylalanine, tyrosine and tryptophan biosynthesis; alanine, aspartate and glutamate metabolism; glycine, serine and threonine metabolism) were selected as important metabolic pathways for hindlimb unloading effect. However, LIPUS promoted the stability of alanine, aspartate and glutamate metabolism pathway. These results suggest that the key mechanism of LIPUS in preventing muscle atrophy induced by hindlimb unloading may be related to promoting protein synthesis through MSTN/Akt/mTOR signaling pathway and stabilizing alanine, aspartate and glutamate metabolism.

Scalable Microgravity Simulator Used for Long-Term Musculoskeletal Cells and Tissue Engineering.

We introduce a new benchtop microgravity simulator (MGS) that is scalable and easy to use. Its working principle is similar to that of random positioning machines (RPM), commonly used in research laboratories and regarded as one of the gold standards for simulating microgravity. The improvement of the MGS concerns mainly the algorithms controlling the movements of the samples and the design that, for the first time, guarantees equal treatment of all the culture flasks undergoing simulated microgravity. Qualification and validation tests of the new device were conducted with human bone marrow stem cells (bMSC) and mouse skeletal muscle myoblasts (C2C12). bMSC were cultured for 4 days on the MGS and the RPM in parallel. In the presence of osteogenic medium, an overexpression of osteogenic markers was detected in the samples from both devices. Similarly, C2C12 cells were maintained for 4 days on the MGS and the rotating wall vessel (RWV) device, another widely used microgravity simulator. Significant downregulation of myogenesis markers was observed in gravitationally unloaded cells. Therefore, similar results can be obtained regardless of the used simulated microgravity devices, namely MGS, RPM, or RWV. The newly developed MGS device thus offers easy and reliable long-term cell culture possibilities under simulated microgravity conditions. Currently, upgrades are in progress to allow real-time monitoring of the culture media and liquids exchange while running. This is of particular interest for long-term cultivation, needed for tissue engineering applications. Tissue grown under real or simulated microgravity has specific features, such as growth in three-dimensions (3D). Growth in weightlessness conditions fosters mechanical, structural, and chemical interactions between cells and the extracellular matrix in any direction.

Fine Tuning of Calcium Constitutive Entry by Optogenetically-Controlled Membrane Polarization: Impact on Cell Migration.

Anomalies in constitutive calcium entry (CCE) have been commonly attributed to cell dysfunction in pathological conditions such as cancer. Calcium influxes of this type rely on channels, such as transient receptor potential (TRP) channels, to be constitutively opened and strongly depend on membrane potential and a calcium driving force. We developed an optogenetic approach based on the expression of the halorhodopsin chloride pump to study CCE in non-excitable cells. Using C2C12 cells, we found that halorhodopsin can be used to achieve a finely tuned control of membrane polarization. Escalating the membrane polarization by incremental changes in light led to a concomitant increase in CCE through transient receptor potential vanilloid 2 (TRPV2) channels. Moreover, light-induced calcium entry through TRPV2 channels promoted cell migration. Our study shows for the first time that by modulating CCE and related physiological responses, such as cell motility, halorhodopsin serves as a potentially powerful tool that could open new avenues for the study of CCE and associated cellular behaviors.

Effect of Photobiomodulation on C2C12 Myoblasts Cultivated in M1 Macrophage-conditioned Media.

Moderate levels of a proinflammatory macrophages phenotype are indispensable and play an important role in the skeletal muscle repair process since this response depends on their secreted products concentration to influence and modulate muscle inflammation as well as the differentiation of myoblasts. This study investigated the effects of photobiomodulation (PBM) on undifferentiated and differentiation-induced C2C12 myoblasts cultivated in different concentrations of M1 phenotype macrophage-conditioned media of J774 cells (MCM1) also submitted to PBM using the same irradiation parameters. Irradiation was performed once with low-level laser (780 nm, 70 mW, 1 J) and was evaluated cell viability, proliferation and differentiation, nitric oxide (NO) synthesis and IL-6 and TNF-α protein levels 24 and 48 h after C2C12 irradiation. PBM treatment in undifferentiated myoblasts exhibited lower IL-6 levels in the presence of nonirradiated MCM1 at both concentrations. Myoblasts in proliferation condition cultivated with irradiated MCM1 showed lower IL-6 and TNF-α levels after 48 h in the presence of both concentrations evaluated. PBM induced a decrease in the synthesis of NO on undifferentiated and differentiation-induced myoblasts. PBM was able to reduce the level of proinflammatory protein and markers, which are important to allow the differentiation of myoblasts during the muscle repair process.

Characterization of C2C12 cells in simulated microgravity: Possible use for myoblast regeneration.

Muscle loss is a major problem for many in lifetime. Muscle and bone degeneration has also been observed in individuals exposed to microgravity and in unloading conditions. C2C12 myoblst cells are able to form myotubes, and myofibers and these cells have been employed for muscle regeneration purposes and in myogenic regeneration and transplantation studies. We exposed C2C12 cells in an random position machine to simulate microgravity and study the energy and the biochemical challenges associated with this treatment. Simulated microgravity exposed C2C12 cells maintain positive proliferation indices and delay the differentiation process for several days. On the other hand this treatment significantly alters many of the biochemical and the metabolic characteristics of the cell cultures including calcium homeostasis. Recent data have shown that these perturbations are due to the inhibition of the ryanodine receptors on the membranes of intracellular calcium stores. We were able to reverse this perturbations treating cells with thapsigargin which prevents the segregation of intracellular calcium ions in the mitochondria and in the sarco/endoplasmic reticula. Calcium homeostasis appear a key target of microgravity exposure. In conclusion, in this study we reported some of the effects induced by the exposure of C2C12 cell cultures to simulated microgravity. The promising information obtained is of fundamental importance in the hope to employ this protocol in the field of regenerative medicine.

The Late Effects of Radiation Therapy on Skeletal Muscle Morphology and Progenitor Cell Content are Influenced by Diet-Induced Obesity and Exercise Training in Male Mice.

Radiation exposure during muscle development induces long-term decrements to skeletal muscle health, which contribute to reduced quality of life in childhood cancer survivors. Whether the effects of radiation on skeletal muscle are influenced by relevant physiological factors, such as obesity and exercise training remains unknown. Using skeletal muscle from our previously published work examining the effects of obesity and exercise training on radiation-exposed bone marrow, we evaluated the influence of these physiological host factors on irradiated skeletal muscle morphology and cellular dynamics. Mice were divided into control and high fat diet groups with or without exercise training. All mice were then exposed to radiation and continued in their intervention group for an additional 4 weeks. Diet-induced obesity resulted in increased muscle fibrosis, while obesity and exercise training both increased muscle adiposity. Exercise training enhanced myofibre cross-sectional area and the number of satellite cells committed to the myogenic lineage. High fat groups demonstrated an increase in p-NFĸB expression, a trend for a decline in IL-6, and increase in TGFB1. These findings suggest exercise training improves muscle morphology and satellite cell dynamics compared to diet-induced obesity in irradiated muscle, and have implications for exercise interventions in cancer survivors.

Low-level laser irradiation induces a transcriptional myotube-like profile in C2C12 myoblasts.

Low-level laser irradiation (LLLI) has been used as a non-invasive method to improve muscular regeneration capability. However, the molecular mechanisms by which LLLI exerts these effects remain largely unknown. Here, we described global gene expression profiling analysis in C2C12 myoblasts after LLLI that identified 514 differentially expressed genes (DEG). Gene ontology and pathway analysis of the DEG revealed transcripts among categories related to cell cycle, ribosome biogenesis, response to stress, cell migration, and cell proliferation. We further intersected the DEG in C2C12 myoblasts after LLLI with publicly available transcriptomes data from myogenic differentiation studies (myoblasts vs myotube) to identify transcripts with potential effects on myogenesis. This analysis revealed 42 DEG between myoblasts and myotube that intersect with altered genes in myoblasts after LLLI. Next, we performed a hierarchical cluster analysis with this set of shared transcripts that showed that LLLI myoblasts have a myotube-like profile, clustering away from the myoblast profile. The myotube-like transcriptional profile of LLLI myoblasts was further confirmed globally considering all the transcripts detected in C2C12 myoblasts after LLLI, by bi-dimensional clustering with myotubes transcriptional profiles, and by the comparison with 154 gene sets derived from previous published in vitro omics data. In conclusion, we demonstrate for the first time that LLLI regulates a set of mRNAs that control myoblast proliferation and differentiation into myotubes. Importantly, this set of mRNAs revealed a myotube-like transcriptional profile in LLLI myoblasts and provide new insights to the understanding of the molecular mechanisms underlying the effects of LLLI on skeletal muscle cells.

Influence of static magnetic fields on human myoblast/mesenchymal stem cell co­cultures.

The results of surgical repair of extensive muscle tissue defects are still of primary concern, leaving patients with residual cosmetic and functional impairments. Therefore, skeletal muscle tissue engineering attempts to grow functional neo­tissue from human stem cells to promote tissue regeneration and support defect closure. Despite intensive research efforts, the goal of stable induction of myogenic differentiation in expanded human stem cells by using clinically feasible stimuli, has not yet been reached to a sufficient extent. Therefore, the present study investigated the differentiation potential of static magnetic fields (SMFs), using co­cultures of human satellite cells and human mesenchymal stem cells (MSCs). It has previously been demonstrated that SMFs may act as a promising myogenic stimulus. Tests were performed on co­cultures with and without SMF exposure, using growth medium [high growth factor concentrations (GM)] and differentiation medium [low growth factors concentrations (DM)]. AlamarBlue® assay­based cell proliferation analysis revealed no significant difference between co­cultures with, vs. without SMF stimulation, regardless of growth factor concentrations in the cell culture medium. To determine the degree of differentiation in co­cultures under stimulation with SMFs, semi­quantitative gene expression measurements of the following marker genes were performed: Desmin, myogenic factor 5, myogenic differentiation antigen 1, myogenin, adult myosin heavy chain 1 and skeletal muscle α1 actin. In neither GM nor DM was a steady, significant increase in marker gene expression detected. Verifying the gene expression findings, immunohistochemical antibody staining against differentiation markers revealed that SMF exposure did not enhance myogenic maturation. Therefore, SMF treatment of human satellite cell/MSC co­cultures did not result in the desired increase in myogenic differentiation. Further studies are required to identify a suitable stimulus for skeletal muscle tissue engineering.

The effect of low intensity shockwave treatment (Li-SWT) on human myoblasts and mouse skeletal muscle.

BACKGROUND: Transplanting myogenic cells and scaffolds for tissue engineering in skeletal muscle have shown inconsistent results. One of the limiting factors is neovascularization at the recipient site. Low intensity shockwave therapy (Li-SWT) has been linked to increased tissue regeneration and vascularization, both integral to survival and integration of transplanted cells. This study was conducted to demonstrate the response of myoblasts and skeletal muscle to Li-SWT. METHOD: Primary isolated human myoblasts and explants were treated with low intensity shockwaves and subsequently cell viability, proliferation and differentiation were tested. Cardiotoxin induced injury was created in tibialis anterior muscles of 28 mice, and two days later, the lesions were treated with 500 impulses of Li-SWT on one of the legs. The treatment was repeated every third day of the period and ended on day 14 after cardiotoxin injection.. The animals were followed up and documented up to 21 days after cardiotoxin injury. RESULTS: Li-SWT had no significant effect on cell death, proliferation, differentiation and migration, the explants however showed decreased adhesion. In the animal experiments, qPCR studies revealed a significantly increased expression of apoptotic, angiogenic and myogenic genes; expression of Bax, Bcl2, Casp3, eNOS, Pax7, Myf5 and Met was increased in the early phase of regeneration in the Li-SWT treated hind limbs. Furthermore, a late accumulative angiogenic effect was demonstrated in the Li-SWT treated limbs by a significantly increased expression of Angpt1, eNOS, iNOS, Vegfa, and Pecam1. CONCLUSION: Treatment was associated with an early upregulation in expression of selected apoptotic, pro-inflammatory, angiogenic and satellite cell activating genes after muscle injury. It also showed a late incremental effect on expression of pro-angiogenic genes. However, we found no changes in the number of PAX7 positive cells or blood vessel density in Li-SWT treated and control muscle. Furthermore, Li-SWT in the selected doses did not decrease survival, proliferation or differentiation of myoblasts in vitro.

White light emitting diode suppresses proliferation and induces apoptosis in hippocampal neuron cells through mitochondrial cytochrome c oxydase-mediated IGF-1 and TNF-α pathways.

Light emitting diode (LED) light has been tested to treat traumatic brain injury, neural degenerative diseases and psychiatric disorders. Previous studies indicate that blue LED light affects cell proliferation and apoptosis in photosensitive cells and cancer cells. In this study, we demonstrate that white LED light exposure impaired proliferation and induced apoptosis in HeLa and HT-22 hippocampal neural cells, but not C2C12 cells. Furthermore, the mechanisms underlying the effect of white LED light exposure on HT-22 cells were elucidated. In HeLa and HT-22 cells, white LED light activated mitochondrial cytochrome c oxidase (Cco), in association with enhanced ATP synthase activity and elevated intracellular ATP concentration. Also, reactive oxygen species (ROS) and nitric oxide (NO) production were increased, accompanied by higher calcium concentration and lower mitochondrial membrane potential. HT-22 cells exposed to white LED light for 24h showed reduced viability, with higher apoptotic rate and a cell cycle arrest at G0/G1 phase. Concurrently, the mRNA expression and the concentration of IGF-1 were decreased, while that of TNF-α were increased, in light-exposed cells, which was supported by the luciferase activity of both gene promoters. The down-stream mitogen-activated protein kinase (MAPK), AKT/mTOR pathways were inhibited, in association with an activation of apoptotic caspase 3. N-Acetylcysteine, a ROS scavenger, protected the cells from LED light-induced cellular damage, with rescued cell viability and restored mRNA expression of IGF-1 and TNF-α. Our data demonstrate that white LED light suppresses proliferation and induces apoptosis in hippocampal neuron cells through mitochondrial Cco/ROS-mediated IGF-1 and TNF-α pathways.

Extremely Low-Frequency Electromagnetic Fields Affect Myogenic Processes in C2C12 Myoblasts: Role of Gap-Junction-Mediated Intercellular Communication.

Extremely low-frequency electromagnetic fields (ELF-EMFs) can interact with biological systems. Although they are successfully used as therapeutic agents in physiatrics and rehabilitative practice, they might represent environmental pollutants and pose a risk to human health. Due to the lack of evidence of their mechanism of action, the effects of ELF-EMFs on differentiation processes in skeletal muscle were investigated. C2C12 myoblasts were exposed to ELF-EMFs generated by a solenoid. The effects of ELF-EMFs on cell viability and on growth and differentiation rates were studied using colorimetric and vital dye assays, cytomorphology, and molecular analysis of MyoD and myogenin expression, respectively. The establishment of functional gap junctions was investigated analyzing connexin 43 expression levels and measuring cell permeability, using microinjection/dye-transfer assays. The ELF-EMFs did not affect C2C12 myoblast viability or proliferation rate. Conversely, at ELF-EMF intensity in the mT range, the myogenic process was accelerated, through increased expression of MyoD, myogenin, and connexin 43. The increase in gap-junction function suggests promoting cell fusion and myotube differentiation. These data provide the first evidence of the mechanism through which ELF-EMFs may provide therapeutic benefits and can resolve, at least in part, some conditions of muscle dysfunction.

Influence of Nitric Oxide generated through microwave plasma on L6 skeletal muscle cell myogenesis via oxidative signaling pathways.

Myogenic precursors are myoblasts that have a potency to differentiate into muscle fibers on injury and maintain the regenerative power of skeletal muscle. However, the roles of exogenous nitric oxide (NO) in muscle development and myoblast differentiation are largely unknown. Therefore, in this study, we examined the effects of exogenous NO generated by a microwave plasma torch on rat myoblastic L6 cell proliferation and differentiation. We observed that the differentiation of L6 myogenic precursor cells into myotubes was significantly enhanced after NO treatment. The expression of the myogenesis marker proteins and mRNA level, such as myoD, myogenin, and myosin heavy chain (MHC), as well as the cyclic guanosine monophosphate (cGMP) level, were significantly increased after the NO treatment, without creating toxicity. Moreover, we observed that the oxidative stress signaling [extracellular-signal-regulated kinase (Erks), and Adenosine monophosphate-activated protein kinase (AMPK)] phosphorylation was higher in NO treated cells than in the control cells [without NO treatment]. Therefore, these results reveal the exogenous NO role in regulating myoblast differentiation through the oxidative stress signaling pathway. Through this work, we can suggest that exogenous NO can help in cell differentiation and tissue regeneration, which provides new possibilities for plasma medicine.

Role of pulsed electromagnetic fields (PEMF) on tenocytes and myoblasts-potential application for treating rotator cuff tears.

The post-surgery integrity of the tendons and muscle quality are the two major factors in success of rotator cuff (RC) repair. Though surgical techniques for rotator cuff repair have significantly improved in the past two decades, there are no effective treatments to improve tendon-to-bone healing and muscle quality after repair at this point in time. Pulsed electromagnetic fields (PEMF) have previously been used for promoting fracture healing. Previous studies have shown that PEMF has a positive role in promoting osteoblast precursors proliferation and differentiation. However, PEMFs effect on tenocytes and muscle cells has not been determined fully yet. The purpose of this study is to define the role of a commercially available PEMF on tenocytes and myoblasts growth and differentiation in vitro. Human rotator cuff tenocytes and C2C12 murine myoblasts were cultured and treated with PEMF for 2 weeks under regular and inflammatory conditions. Our results showed that 2 weeks treatment of PEMF enhanced gene expressions of growth factors in human rotator cuff tenocytes under inflammatory conditions. PEMF significantly enhanced C2C12 myotube formation under normal and inflammatory conditions. Results from this study suggest that PEMF has a positive role in promoting tenocyte gene expression and myoblast differentiation. Therefore, PEMF may potentially serve as a non-operative treatment to improve clinical incomes rotator cuff tendon repairs. Results © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:956-964, 2017.

Low-level infrared laser modulates muscle repair and chromosome stabilization genes in myoblasts.

Infrared laser therapy is used for skeletal muscle repair based on its biostimulative effect on satellite cells. However, shortening of telomere length limits regenerative potential in satellite cells, which occurs after each cell division cycle. Also, laser therapy could be more effective on non-physiologic tissues. This study evaluated low-level infrared laser exposure effects on mRNA expression from muscle injury repair and telomere stabilization genes in myoblasts in normal and stressful conditions. Laser fluences were those used in clinical protocols. C2C12 myoblast cultures were exposed to low-level infrared laser (10, 35, and 70 J/cm(2)) in standard or normal (10 %) and reduced (2 %) fetal bovine serum concentrations; total RNA was extracted for mRNA expression evaluation from muscle injury repair (MyoD and Pax7) and chromosome stabilization (TRF1 and TRF2) genes by real time quantitative polymerization chain reaction. Data show that low-level infrared laser increases the expression of MyoD and Pax7 in 10 J/cm(2) fluence, TRF1 expression in all fluences, and TRF2 expression in 70 J/cm(2) fluence in both 10 and 2 % fetal bovine serum. Low-level infrared laser increases mRNA expression from genes related to muscle repair and telomere stabilization in myoblasts in standard or normal and stressful conditions.

Photobiomodulation Protects and Promotes Differentiation of C2C12 Myoblast Cells Exposed to Snake Venom.

BACKGROUND: Snakebites is a neglected disease and in Brazil is considered a serious health problem, with the majority of the snakebites caused by the genus Bothrops. Antivenom therapy and other first-aid treatments do not reverse local myonecrose which is the main sequel caused by the envenomation. Several studies have shown the effectiveness of low level laser (LLL) therapy in reducing local myonecrosis induced by Bothropic venoms, however the mechanism involved in this effect is unknown. In this in vitro study, we aimed to analyze the effect of LLL irradiation against cytotoxicity induced by Bothrops jararacussu venom on myoblast C2C12 cells. METHODOLOGY: C2C12 were utilized as a model target and were incubated with B. jararacussu venom (12.5 µg/mL) and immediately irradiated with LLL at wavelength of red 685 nm or infrared 830 nm with energy density of 2.0, 4.6 and 7.0 J/cm2. Effects of LLL on cellular responses of venom-induced cytotoxicity were examined, including cell viability, measurement of cell damage and intra and extracellular ATP levels, expression of myogenic regulatory factors, as well as cellular differentiation. RESULTS: In non-irradiated cells, the venom caused a decrease in cell viability and a massive release of LDH and CK levels indicating myonecrosis. Infrared and red laser at all energy densities were able to considerably decrease venom-induced cytotoxicity. Laser irradiation induced myoblasts to differentiate into myotubes and this effect was accompanied by up regulation of MyoD and specially myogenin. Moreover, LLL was able to reduce the extracellular while increased the intracellular ATP content after venom exposure. In addition, no difference in the intensity of cytotoxicity was shown by non-irradiated and irradiated venom. CONCLUSION: LLL irradiation caused a protective effect on C2C12 cells against the cytotoxicity caused by B. jararacussu venom and promotes differentiation of these cells by up regulation of myogenic factors. A modulatory effect of ATP synthesis may be suggested as a possible mechanism mediating cytoprotection observed under laser irradiation.

Oxidative Stress and Plasma Membrane Repair in Single Myoblasts After Femtosecond Laser Photoporation.

Cell membranes are susceptible to biophysical damages. These biophysical damages often present themselves in challenging oxidative environments, such as in chronic inflammation. Here we report the damage evolution after single myoblasts were individually subjected to femtosecond (fs) laser photoporation on their plasma membranes under normal and oxidative conditions. A well-characterized tunable fs laser was coupled with a laser scanning confocal microscope. The post-damage wound evolution was documented by real-time imaging. The fs laser could generate a highly focused hole at a targeted site of the myoblast plasma membrane. The initial hole size depended on the laser dosage in terms of power and exposure duration. With the same laser power and irradiation duration, photoporation invoked bigger holes in the oxidative groups than in the control. Myoblasts showed difficulty in repairing holes with initial size beyond certain threshold. Within the threshold, holes could apparently be resealed within 100 s under the normal condition; while in oxidative condition, the resealing process could take 100-300 s. The hole-resealing capacity of myoblasts was compromised under oxidative stress particularly when the oxidative exposure was chronic. It is interesting to note that brief exposure to oxidative stress apparently could promote resealing in myoblasts after photoporation.

Low-level laser irradiation modulates cell viability and creatine kinase activity in C2C12 muscle cells during the differentiation process.

Low-level laser irradiation (LLLI) is increasingly used to treat musculoskeletal disorders, with satisfactory results described in the literature. Skeletal muscle satellite cells play a key role in muscle regeneration. The aim of the present study was to evaluate the effect of LLLI on cell viability, creatine kinase (CK) activity, and the expression of myogenic regulatory factors in C2C12 myoblasts during the differentiation process. C2C12 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 2% horse serum and submitted to irradiation with GaAlAs diode laser (wavelength, 780 nm; output power, 10 mW; energy density, 5 J/cm2). Cell viability and the expression of myogenic regulatory factors were assessed 24, 48, and 72 h after irradiation by 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyltetrazolium bromide (MTT) assay and quantitative real-time polymerase chain reaction (RT-qPCR), respectively. CK activity was analyzed at 24 and 72 h. An increase in cell viability was found in the laser group in comparison to the control group at all evaluation times. CK activity was significantly increased in the laser group at 72 h. Myogenin messenger RNA (mRNA) demonstrated a tendency toward an increase in the laser group, but the difference in comparison to the control group was non-significant. In conclusion, LLLI was able to modulate cell viability and CK activity in C2C12 myoblasts during the differentiation process.

Chitosan selectively promotes adhesion of myoblasts over fibroblasts.

Treatment of ventral hernias frequently makes use of synthetic and biological meshes, often resulting in scar tissue formation and incomplete muscle regeneration. Chitosan has been shown to promote a regenerative response than a fibrotic response. The objective of this study is to determine the effects of chitosan on myoblast and fibroblast adhesion and the underlying mechanisms. Primary myoblast cells, embryonic fibroblasts, and 1:1 coculture of both cell types were seeded on chitosan-coated and uncoated tissue culture plates. The number of attached cells was measured at 5, 24, 48, and 72 h after seeding. At 5 h after seeding, expression and structural organization of actin, integrin ß3, and integrin ß1 were analyzed. With chitosan, primary fibroblasts had 2.7 times smaller F-actin fraction, and more primary myoblasts with higher expression of integrin ß3 were seen. Moreover, a higher ratio of myoblasts to fibroblasts was seen with chitosan, with a ratio higher than one at 48 and 72 h after seeding and greater than a twofold increase in ratio when compared with uncoated plates. A higher organization of actin and integrin ß3 network around nucleus of myoblasts was observed with chitosan, whereas reorganization in actin and integrin ß1 network was observed in fibroblasts. In conclusion, chitosan promotes myoblast adhesion with higher expression of integrin ß3 and inhibits fibroblast adhesion with reorganization of actin and integrin ß1 network.

VBP15, a novel anti-inflammatory and membrane-stabilizer, improves muscular dystrophy without side effects.

Absence of dystrophin makes skeletal muscle more susceptible to injury, resulting in breaches of the plasma membrane and chronic inflammation in Duchenne muscular dystrophy (DMD). Current management by glucocorticoids has unclear molecular benefits and harsh side effects. It is uncertain whether therapies that avoid hormonal stunting of growth and development, and/or immunosuppression, would be more or less beneficial. Here, we discover an oral drug with mechanisms that provide efficacy through anti-inflammatory signaling and membrane-stabilizing pathways, independent of hormonal or immunosuppressive effects. We find VBP15 protects and promotes efficient repair of skeletal muscle cells upon laser injury, in opposition to prednisolone. Potent inhibition of NF-κB is mediated through protein interactions of the glucocorticoid receptor, however VBP15 shows significantly reduced hormonal receptor transcriptional activity. The translation of these drug mechanisms into DMD model mice improves muscle strength, live-imaging and pathology through both preventive and post-onset intervention regimens. These data demonstrate successful improvement of dystrophy independent of hormonal, growth, or immunosuppressive effects, indicating VBP15 merits clinical investigation for DMD and would benefit other chronic inflammatory diseases.