Comparison of Three Antagonists of Hedgehog Pathway to Promote Skeletal Muscle Regeneration after High Dose Irradiation.

The current geopolitical context has brought the radiological nuclear risk to the forefront of concerns. High-dose localized radiation exposure leads to the development of a musculocutaneous radiation syndrome affecting the skin and subcutaneous muscles. Despite the implementation of a gold standard treatment based on an invasive surgical procedure coupled with autologous cell therapy, a muscular defect frequently persists. Targeting the modulation of the Hedgehog (Hh) signaling pathway appears to be a promising therapeutic approach. Activation of this pathway enhances cell survival and promotes proliferation after irradiation, while inhibition by Cyclopamine facilitates differentiation. In this study, we compared the effects of three antagonists of Hh, Cyclopamine (CA), Vismodegib (VDG) and Sonidegib (SDG) on differentiation. A stable cell line of murine myoblasts, C2C12, was exposed to X-ray radiation (5 Gy) and treated with CA, VDG or SDG. Analysis of proliferation, survival (apoptosis), morphology, myogenesis genes expression and proteins production were performed. According to the results, VDG does not have a significant impact on C2C12 cells. SDG increases the expression/production of differentiation markers to a similar extent as CA, while morphologically, SDG proves to be more effective than CA. To conclude, SDG can be used in the same way as CA but already has a marketing authorization with an indication against basal cell cancers, facilitating their use in vivo. This proof of concept demonstrates that SDG represents a promising alternative to CA to promotes differentiation of murine myoblasts. Future studies on isolated and cultured satellite cells and in vivo will test this proof of concept.

DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood. Recently, we demonstrated the overexpression of both DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B) in RMS tumour biopsies and cell lines compared to normal skeletal muscle. Radiotherapy may often fail due to the abnormal expression of some molecules able to drive resistance mechanisms. The aim of this study was to analyse the involvement of DNMT3A and DNMT3B in radioresistance in RMS. RNA interference experiments against DNMT3A/3B were performed in embryonal RMS cells, upon ionizing radiation (IR) exposure and the effects of the combined treatment on RMS cells were analysed. DNMT3A and DNMT3B knocking down increased the sensitivity of RMS cells to IR, as indicated by the drastic decrease of colony formation ability. Interestingly, DNMT3A/3B act in two different ways: DNMT3A silencing triggers the cellular senescence program by up-regulating p16 and p21, whilst DNMT3B depletion induces significant DNA damage and impairs the DNA repair machinery (ATM, DNA-PKcs and Rad51 reduction). Our findings demonstrate for the first time that DNMT3A and DNMT3B overexpression may contribute to radiotherapy failure, and their inhibition might be a promising radiosensitizing strategy, mainly in the treatment of patients with metastatic or recurrent RMS tumours.

Mechanical Stimulation of Adhesion Receptors Using Light-Responsive Nanoparticle Actuators Enhances Myogenesis.

The application of cyclic strain is known to enhance myoblast differentiation and muscle growth in vitro and in vivo. However, current techniques apply strain to full tissues or cell monolayers, making it difficult to evaluate whether mechanical stimulation at the subcellular or single-cell scales would drive myoblast differentiation. Here, we report the use of optomechanical actuator (OMA) particles, comprised of a ∼0.6 µm responsive hydrogel coating a gold nanorod (100 × 20 nm) core, to mechanically stimulate the integrin receptors in myoblasts. When illuminated with near-infrared (NIR) light, OMA nanoparticles rapidly collapse, exerting mechanical forces to cell receptors bound to immobilized particles. Using a pulsed illumination pattern, we applied cyclic integrin forces to C2C12 myoblasts cultured on a monolayer of OMA particles and then measured the cellular response. We found that 20 min of OMA actuation resulted in cellular elongation in the direction of the stimulus and enhancement of nuclear YAP1 accumulation, an effector of ERK phosphorylation. Cellular response was dependent on direct conjugation of RGD peptides to the OMA particles. Repeated OMA mechanical stimulation for 5 days led to enhanced myogenesis as quantified using cell alignment, fusion, and sarcomeric myosin expression in myotubes. OMA-mediated myogenesis was sensitive to the geometry of stimulation but not to MEK1/2 inhibition. Finally, we found that OMA stimulation in regions proximal to the nucleus resulted in localization of the transcription activator YAP-1 to the nucleus, further suggesting the role of YAP1 in mechanotransduction in C2C12 cells. These findings demonstrate OMAs as a novel tool for studying the role of spatially localized forces in influencing myogenesis.

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.

A systems biology approach reveals neuronal and muscle developmental defects after chronic exposure to ionising radiation in zebrafish.

Contamination of the environment after the Chernobyl and Fukushima Daiichi nuclear power plant (NPP) disasters led to the exposure of a large number of humans and wild animals to radioactive substances. However, the sub-lethal consequences induced by these absorbed radiological doses remain understudied and the long-term biological impacts largely unknown. We assessed the biological effects of chronic exposure to ionizing radiation (IR) on embryonic development by exposing zebrafish embryo from fertilization and up to 120 hours post-fertilization (hpf) at dose rates of 0.5 mGy/h, 5 mGy/h and 50 mGy/h, thereby encompassing the field of low dose rates defined at 6 mGy/h. Chronic exposure to IR altered larval behaviour in a light-dark locomotor test and affected cardiac activity at a dose rate as low as 0.5 mGy/h. The multi-omics analysis of transcriptome, proteome and transcription factor binding sites in the promoters of the deregulated genes, collectively points towards perturbations of neurogenesis, muscle development, and retinoic acid (RA) signaling after chronic exposure to IR. Whole-mount RNA in situ hybridization confirmed the impaired expression of the transcription factors her4.4 in the central nervous system and myogenin in the developing muscles of exposed embryos. At the organ level, the assessment of muscle histology by transmission electron microscopy (TEM) demonstrated myofibers disruption and altered neuromuscular junctions in exposed larvae at 5 mGy/h and 50 mGy/h. The integration of these multi-level data demonstrates that chronic exposure to low dose rates of IR has an impact on neuronal and muscle progenitor cells, that could lead to motility defects in free swimming larvae at 120 hpf. The mechanistic understanding of these effects allows us to propose a model where deregulation of RA signaling by chronic exposure to IR has pleiotropic effects on neurogenesis and muscle development.

Photobiomodulation and different macrophages phenotypes during muscle tissue repair.

Macrophages play a very important role in the conduction of several regenerative processes mainly due to their plasticity and multiple functions. In the muscle repair process, while M1 macrophages regulate the inflammatory and proliferative phases, M2 (anti-inflammatory) macrophages direct the differentiation and remodelling phases, leading to tissue regeneration. The aim of this study was to evaluate the effect of red and near infrared (NIR) photobiomodulation (PBM) on macrophage phenotypes and correlate these findings with the repair process following acute muscle injury. Wistar rats were divided into 4 groups: control; muscle injury; muscle injury + red PBM; and muscle injury + NIR PBM. After 2, 4 and 7 days, the tibialis anterior muscle was processed for analysis. Macrophages phenotypic profile was evaluated by immunohistochemistry and correlated with the different stages of the skeletal muscle repair by the qualitative and quantitative morphological analysis as well as by the evaluation of IL-6, TNF-α and TGF-ß mRNA expression. Photobiomodulation at both wavelengths was able to decrease the number of CD68+ (M1) macrophages 2 days after muscle injury and increase the number of CD163+ (M2) macrophages 7 days after injury. However, only NIR treatment was able to increase the number of CD206+ M2 macrophages (Day 2) and TGF-ß mRNA expression (Day 2, 4 and 7), favouring the repair process more expressivelly. Treatment with PBM was able to modulate the inflammation phase, optimize the transition from the inflammatory to the regeneration phase (mainly with NIR light) and improve the final step of regeneration, enhancing tissue repair.

Promotion of Myogenic Maturation by Timely Application of Electric Field Along the Topographical Alignment.

Engineered muscular substitutes can restore the impaired muscle functions when integrated properly into the host tissue. To generate functional muscles with sufficient contractility at the site of transplant, the in vitro construction of fully differentiated muscle fibers would be desired. Many previous reports have identified either topographical alignment or electrical stimulation as an effective tool to promote myogenic differentiation. However, optimization of spatial and temporal arrangement of these two physical cues for better differentiation and maturation of skeletal muscles has not been investigated. In this article, we introduce a novel cell culture system that allows simultaneous application of these two independent directional cues at both orthogonal and parallel arrangements. We then show that the parallel arrangement of the aligned topography and the electric field synergistically facilitates better differentiation and maturation of C2C12, generating myotubes with more fused nuclei. Addition of the electric stimulation at the late stage of myogenic differentiation is found to further improve cell fusion to form multinucleate myotubes through a phosphatidylinositol-3-OH-kinase-dependent pathway. As such, we successfully demonstrated that the combined stimulation of topographical and electrical cues could effectively enhance both myogenic differentiation and maturation in a temporal and orientation-dependent manner, providing the basis for therapeutic strategies for regenerative 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.

Various LED Wavelengths Affected Myofiber Development and Satellite Cell Proliferation of Chick Embryos via the IGF-1 Signaling Pathway.

An effect of monochromatic light illumination on muscle mass has been discovered in chickens; however, its effect on the development of embryonic muscle remains unclear. Our previous studies demonstrated that monochromatic green light promoted satellite cell proliferation and muscle growth in posthatching broilers. In this study, we investigated the effects and mechanisms of monochromatic light exposure on muscle development in late embryogenesis. Seven hundred and fifty fertile broiler eggs were randomly assigned to blue (B-group), green (G-group), red (R-group), white (W-group) lights or darkness (D-group) throughout the incubation period. The muscle weight and fiber size were highest in the G-group compared to the other groups during embryonic days (E) 17 to E20. The proliferation of satellite cells isolated from the G-group was highest, and in vivo green light remarkably increased the number of proliferating cell nuclear antigen (PCNA)-positive cells in skeletal muscle. Meanwhile, plasma IGF-1 was higher (15.5-16.2%) in the G-group than that in D- and R-groups, and the satellite cells isolated from the G-group had a more sensitive response to IGF-1. These findings demonstrate green monochromatic photobiomodulation promoted the muscle growth and satellite cell proliferation was related to the IGF-1 signaling pathway in late embryogenesis.

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.

In ovo exposure to monochromatic lights affect posthatch muscle growth and satellite cell proliferation of chicks: role of IGF-1.

To study the role of IGF-1 on stimulation with monochromatic light during incubation altering posthatch muscle growth, chicken embryos were exposed to blue light, green light, red light, white light or darkness throughout embryonic period and then were raised in white light conditions upon hatching. Comparing with the other treatment groups, the chicks in green light group had heavier hatching weights, higher muscle indexes and larger muscle fibers. Both in vivo and in vitro studies showed that the number and proliferative activity of satellite cells in green light group were the highest. Plasma IGF-1 level and skeletal muscle IGF-1R mRNA level were higher in green light group. Moreover, exogenous IGF-1 increased the proliferative activity of satellite cell in a dose-dependent fashion. These results suggest that stimulation with monochromatic green light during incubation promoted posthatch muscle growth and satellite cell proliferation of chicks through IGF-1 signaling.

Electrically driven intracellular and extracellular nanomanipulators evoke neurogenic/cardiomyogenic differentiation in human mesenchymal stem cells.

Nanomechanical intervention through electroactuation is an effective strategy to guide stem cell differentiation for tissue engineering and regenerative medicine. In the present study, we elucidate that physical forces exerted by electroactuated gold nanoparticles (GNPs) have a strong influence in regulating the lineage commitment of human mesenchymal stem cells (hMSCs). A novel platform that combines intracellular and extracellular GNPs as nano-manipulators was designed to trigger neurogenic/cardiomyogenic differentiation in hMSCs, in electric field stimulated culture condition. In order to mimic the native microenvironment of nerve and cardiac tissues, hMSCs were treated with physiologically relevant direct current electric field (DC EF) or pulsed electric field (PEF) stimuli, respectively. When exposed to regular intermittent cycles of DC EF stimuli, majority of the GNP actuated hMSCs acquired longer filopodial extensions with multiple branch-points possessing neural-like architecture. Such morphological changes were consistent with higher mRNA expression level for neural-specific markers. On the other hand, PEF elicited cardiomyogenic differentiation, which is commensurate with the tube-like morphological alterations along with the upregulation of cardiac specific markers. The observed effect was significantly promoted even by intracellular actuation and was found to be substrate independent. Further, we have substantiated the participation of oxidative signaling, G0/G1 cell cycle arrest and intracellular calcium [Ca(2+)]i elevation as the key upstream regulators dictating GNP assisted hMSC differentiation. Thus, by adopting dual stimulation protocols, we could successfully divert the DC EF exposed cells to differentiate predominantly into neural-like cells and PEF treated cells into cardiomyogenic-like cells, via nanoactuation of GNPs. Such a novel multifaceted approach can be exploited to combat tissue loss following brain injury or heart failure.

Effect of Green Light on Nitric Oxide Metabolism in Chick Embryos. A Possible Physiological Role.

The exposure to green light, which serves as a well-known activating factor for myogenesis during incubation of chicken eggs, contributes to intensification of embryonic metabolism of NO. A metabolic product, nitrate, is mainly accumulated in the muscles. These data suggest that light induces a NO-dependent activation of the factor, which intensifies muscle tissue development.

Redistribution of body composition in patients with Graves' disease after iodine-131 treatment.

OBJECTIVE: The objective of this study was to investigate body composition redistribution at 3 months after radioactive iodine therapy (RAI). METHODS: Eighty patients with Graves' disease (GD) for RAI and 18 volunteers were recruited. All patients underwent thyroid status test and dual-energy x-ray absorptiometry at baseline and 3 months after RAI. According to the second thyroid status test, patients were divided into the following groups: A, with aggravated hyperthyroidism; B-1, with improved hyperthyroidism; B-2, with euthyroidism; and B-3, with hypothyroidism. RESULTS: Total lean mass (LM) but fat mass (FM) and bone mineral content (BMC) of whole GD patients after RAI recovered to be not different with controls. Compared with baseline, in group A, FM in the left leg increased, and LM in left arm, right arm, trunk and total LM decreased (P<0.05). In B-2, FM in the head increased, and LM in the head, right arm, trunk and total LM increased (P<0.05). In B-3, FM in the right leg and total body fat percentage decreased, but FM in the head, android-to-gynoid fat ratio and body mass index increased (P<0.05); LM of all sites, weight and total mass increased (P<0.05); BMC in lumbar spine and left leg, and total BMC decreased (P<0.05). Body composition of unmentioned sites was retained after RAI in each group (P>0.05). CONCLUSIONS: Replenishment of LM gets priority rather than FM and BMC during the first 3 months after RAI, and the increase in LM starts from the upper body; head is the regional site in which FM recovery occurs first.

Nonionizing radiation as a noninvasive strategy in regenerative medicine: the effect of Ca(2+)-ICR on mouse skeletal muscle cell growth and differentiation.

Controlling cell differentiation and proliferation with minimal manipulation is one of the most important goals for cell therapy in clinical applications. In this work, we evaluated the hypothesis that the exposure of myoblast cells (C2C12) to nonionizing radiation (tuned at an extremely low-frequency electromagnetic field at calcium-ion cyclotron frequency of 13.75 Hz) may drive their differentiation toward a myogenic phenotype. C2C12 cells exposed to calcium-ion cyclotron resonance (Ca(2+)-ICR) showed a decrease in cellular growth and an increase in the G(0)/G(1) phase. Severe modifications in the shape and morphology and a change in the actin distribution were revealed by the phalloidin fluorescence analysis. A significant upregulation at transcriptional and translational levels of muscle differentiation markers such as myogenin (MYOG), muscle creatine kinase (MCK), and alpha skeletal muscle actin (ASMA) was observed in exposed C2C12 cells. Moreover, the pretreatment with nifedipine (an L-type voltage-gated Ca(2+) channel blocker) led to a reduction of the Ca(2+)-ICR effect. Consequently, it induced a downregulation of the MYOG, MCK, and ASMA mRNA expression affecting adversely the differentiation process. Therefore, our data suggest that Ca(2+)-ICR exposure can upregulate C2C12 differentiation. Although further studies are needed, these results may have important implications in myodegenerative pathology therapies.

Extensive mononuclear infiltration and myogenesis characterize recovery of dysferlin-null skeletal muscle from contraction-induced injuries.

We studied the response of dysferlin-null and control skeletal muscle to large- and small-strain injuries to the ankle dorsiflexors in mice. We measured contractile torque and counted fibers retaining 10-kDa fluorescein dextran, necrotic fibers, macrophages, and fibers with central nuclei and expressing developmental myosin heavy chain to assess contractile function, membrane resealing, necrosis, inflammation, and myogenesis. We also studied recovery after blunting myogenesis with X-irradiation. We report that dysferlin-null myofibers retain 10-kDa dextran for 3 days after large-strain injury but are lost thereafter, following necrosis and inflammation. Recovery of dysferlin-null muscle requires myogenesis, which delays the return of contractile function compared with controls, which recover from large-strain injury by repairing damaged myofibers without significant inflammation, necrosis, or myogenesis. Recovery of control and dysferlin-null muscles from small-strain injury involved inflammation and necrosis followed by myogenesis, all of which were more pronounced in the dysferlin-null muscles, which recovered more slowly. Both control and dysferlin-null muscles also retained 10-kDa dextran for 3 days after small-strain injury. We conclude that dysferlin-null myofibers can survive contraction-induced injury for at least 3 days but are subsequently eliminated by necrosis and inflammation. Myogenesis to replace lost fibers does not appear to be significantly compromised in dysferlin-null mice.

Intrabiliary radiation inhibits smooth muscle formation and biliary duct remodelling after balloon overstretching injury in dogs.

BACKGROUND: Internal metallic stents have been widely used in clinical practice, but a high postoperative restenosis rate limits its application. The purpose of this study was to determine the effect of intrabiliary radiation on muscle formation and biliary duct remodeling after biliary duct balloon injury in dogs. METHODS: Twenty male dogs (15 - 20 kg) were randomly divided into treatment group (n = 10) and control group (n = 10). Balloon overstretching injury was induced using a balloon catheter placed across the biliary duct. Subsequently, a 103Pd radioactive stent was positioned at the target site in each animal in the treatment group, providing the injured biliary duct with a radiation dose of 12.58 x 10(7) Bq. Dogs in the control group received Ni-Ti stents. All the dogs were killed one month after initial injury. The injured sections were dissected free from the dogs, and were processed for histological and morphological study. Cross-sections were stained with hematoxylin-eosin, Masson's trichrome, and Verhoef-van Giesen. Muscle formation area and lumen area were determined using a computer-assisted image analysis system. RESULTS: Compared with the control group, 103Pd radioactive stents significantly reduced muscle formation area (78.3%, P < 0.01), and percentage area of stenosis [control stents: (60.0 +/- 21.6)%, 103Pd radioactive stents: (31.6 +/- 9.5)%]. In addition, in the treatment group, the biliary duct lumen area was significantly larger than that in the control group (P < 0.01). CONCLUSIONS: 103Pd radioactive stents providing a radioactive dose of 12.58 x 10(7) Bq are effective in reducing muscle formation and biliary duct remodeling after balloon overstretching injury.