HIF-1α/MMP-9 Axis Is Required in the Early Phases of Skeletal Myoblast Differentiation under Normoxia Condition In Vitro.

Hypoxia-inducible factor (HIF)-1α represents an oxygen-sensitive subunit of HIF transcriptional factor, which is usually degraded in normoxia and stabilized in hypoxia to regulate several target gene expressions. Nevertheless, in the skeletal muscle satellite stem cells (SCs), an oxygen level-independent regulation of HIF-1α has been observed. Although HIF-1α has been highlighted as a SC function regulator, its spatio-temporal expression and role during myogenic progression remain controversial. Herein, using biomolecular, biochemical, morphological and electrophysiological analyses, we analyzed HIF-1α expression, localization and role in differentiating murine C2C12 myoblasts and SCs under normoxia. In addition, we evaluated the role of matrix metalloproteinase (MMP)-9 as an HIF-1α effector, considering that MMP-9 is involved in myogenesis and is an HIF-1α target in different cell types. HIF-1α expression increased after 24/48 h of differentiating culture and tended to decline after 72 h/5 days. Committed and proliferating mononuclear myoblasts exhibited nuclear HIF-1α expression. Differently, the more differentiated elongated and parallel-aligned cells, which are likely ready to fuse with each other, show a mainly cytoplasmic localization of the factor. Multinucleated myotubes displayed both nuclear and cytoplasmic HIF-1α expression. The MMP-9 and MyoD (myogenic activation marker) expression synchronized with that of HIF-1α, increasing after 24 h of differentiation. By means of silencing HIF-1α and MMP-9 by short-interfering RNA and MMP-9 pharmacological inhibition, this study unraveled MMP-9's role as an HIF-1α downstream effector and the fact that the HIF-1α/MMP-9 axis is essential in morpho-functional cell myogenic commitment.

Vindoline Exhibits Anti-Diabetic Potential in Insulin-Resistant 3T3-L1 Adipocytes and L6 Skeletal Myoblasts.

Type 2 diabetes mellitus (T2DM) emerged as a major health care concern in modern society, primarily due to lifestyle changes and dietary habits. Obesity-induced insulin resistance is considered as the major pathogenic factor in T2DM. In this study, we investigated the effect of vindoline, an indole alkaloid of Catharanthus roseus on insulin resistance (IR), oxidative stress and inflammatory responses in dexamethasone (IR inducer)-induced dysfunctional 3T3-L1 adipocytes and high-glucose-induced insulin-resistant L6-myoblast cells. Results showed that dexamethasone-induced dysfunctional 3T3-L1 adipocytes treated with different concentrations of vindoline significantly enhanced basal glucose consumption, accompanied by increased expression of GLUT-4, IRS-1 and adiponectin. Similarly, vindoline-treated insulin-resistant L6 myoblasts exhibited significantly enhanced glycogen content accompanied with upregulation of IRS-1 and GLUT-4. Thus, in vitro studies of vindoline in insulin resistant skeleton muscle and dysfunctional adipocytes confirmed that vindoline treatment significantly mitigated insulin resistance in myotubes and improved functional status of adipocytes. These results demonstrated that vindoline has the potential to be used as a therapeutic agent to ameliorate obesity-induced T2DM-associated insulin resistance profile in adipocytes and skeletal muscles.

HDAC8 regulates protein kinase D phosphorylation in skeletal myoblasts in response to stress signaling.

Skeletal muscle differentiation involves activation of quiescent satellite cells to proliferate, differentiate and fuse to form new myofibers; this requires coordination of myogenic transcription factors. Myogenic transcription is tightly regulated by various intracellular signaling pathways, which include members of the protein kinase D (PKD) family. PKD is a family of serine-threonine kinases that regulate gene expression, protein secretion, cell proliferation, differentiation and inflammation. PKD is a unique PKC family member that shares distant sequence homology to calcium-regulated kinases and plays an important role in muscle physiology. In this report, we show that class I histone deacetylase (HDAC) inhibition, and in particular HDAC8 inhibition, attenuated PKD phosphorylation in skeletal C2C12 myoblasts in response to phorbol ester, angiotensin II and dexamethasone signaling independent of changes in total PKD protein expression. As class I HDACs and PKD signaling are requisite for myocyte differentiation, these data suggest that HDAC8 functions as a potential feedback regulator of PKD phosphorylation to control myogenic gene expression.

CircRNA Profiling of Skeletal Muscle in Two Pig Breeds Reveals CircIGF1R Regulates Myoblast Differentiation via miR-16.

Muscle development is closely related to meat quality and production. CircRNAs, with a closed-ring structure, have been identified as a key regulator of muscle development. However, the roles and mechanisms of circRNAs in myogenesis are largely unknown. Hence, in order to unravel the functions of circRNAs in myogenesis, the present study explored circRNA profiling in skeletal muscle between Mashen and Large White pigs. The results showed that a total of 362 circRNAs, which included circIGF1R, were differentially expressed between the two pig breeds. Functional assays showed that circIGF1R promoted myoblast differentiation of porcine skeletal muscle satellite cells (SMSCs), while it had no effect on cell proliferation. In consideration of circRNA acting as a miRNA sponge, dual-luciferase reporter and RIP assays were performed and the results showed that circIGF1R could bind miR-16. Furthermore, the rescue experiments showed that circIGF1R could counteract the inhibitory effect of miR-16 on cell myoblast differentiation. Thus, circIGF1R may regulate myogenesis by acting as a miR-16 sponge. In conclusion, this study successfully screened candidate circRNAs involved in the regulation of porcine myogenesis and demonstrated that circIGF1R promotes myoblast differentiation via miR-16, which lays a theoretical foundation for understanding the role and mechanism of circRNAs in regulating porcine myoblast differentiation.

Decrease in the expression of muscle-specific miRNAs, miR-133a and miR-1, in myoblasts with replicative senescence.

Muscles that are injured or atrophied by aging undergo myogenic regeneration. Although myoblasts play a pivotal role in myogenic regeneration, their function is impaired with aging. MicroRNAs (miRNAs) are also involved in myogenic regeneration. MiRNA (miR)-1 and miR-133a are muscle-specific miRNAs that control the proliferation and differentiation of myoblasts. In this study, we determined whether miR-1 and miR-133a expression in myoblasts is altered with cellular senescence and involved in senescence-impaired myogenic differentiation. C2C12 murine skeletal myoblasts were converted to a replicative senescent state by culturing to a high passage number. Although miR-1 and miR-133a expression was largely induced during myogenic differentiation, expression was suppressed in cells at high passage numbers (passage 10 and/or passage 20). Although the senescent myoblasts exhibited a deterioration of myogenic differentiation, transfection of miR-1 or miR-133a into myoblasts ameliorated cell fusion. Treatment with the glutaminase 1 inhibitor, BPTES, removed senescent cells from C2C12 myoblasts with a high passage number, whereas myotube formation and miR-133a expression was increased. In addition, primary cultured myoblasts prepared from aged C57BL/6J male mice (20 months old) exhibited a decrease in miR-1 and miR-133a levels compared with younger mice (3 months old). The results suggest that replicative senescence suppresses muscle-specific miRNA expression in myoblasts, which contributes to the senescence-related dysfunction of myogenic regeneration.

A regenerative niche for stem cells.

Production of hyaluronic acid allows regenerative signaling in muscle stem cells after injury.

JMJD3 activated hyaluronan synthesis drives muscle regeneration in an inflammatory environment.

Muscle stem cells (MuSCs) reside in a specialized niche that ensures their regenerative capacity. Although we know that innate immune cells infiltrate the niche in response to injury, it remains unclear how MuSCs adapt to this altered environment for initiating repair. Here, we demonstrate that inflammatory cytokine signaling from the regenerative niche impairs the ability of quiescent MuSCs to reenter the cell cycle. The histone H3 lysine 27 (H3K27) demethylase JMJD3, but not UTX, allowed MuSCs to overcome inhibitory inflammation signaling by removing trimethylated H3K27 (H3K27me3) marks at the Has2 locus to initiate production of hyaluronic acid, which in turn established an extracellular matrix competent for integrating signals that direct MuSCs to exit quiescence. Thus, JMJD3-driven hyaluronic acid synthesis plays a proregenerative role that allows MuSC adaptation to inflammation and the initiation of muscle repair.

Aqueous extracts of Corni Fructus protect C2C12 myoblasts from DNA damage and apoptosis caused by oxidative stress.

BACKGROUND: Although the various pharmacological effects of Corni Fructus are highly correlated with its antioxidant activity, the blocking effect against oxidative stress in muscle cells is not clear. The purpose of this study was to investigate the effect of aqueous extracts of Corni Fructus (CFE) against oxidative stress caused by hydrogen peroxide (H2O2) in murine skeletal C2C12 myoblasts. METHODS AND RESULTS: MTT assay for cell viability, DCF-DA staining for reactive oxygen species (ROS) production, Comet assay for DNA damage, annexin V-FITC and PI double staining for apoptosis, JC-1 staining and caspase assay for monitor mitochondrial integrity, and western blotting for related protein levels were conducted in H2O2 oxidative stressed C2C12 cells. Our results showed that CFE pretreatment significantly ameliorated the loss of cell viability and inhibited apoptosis in H2O2-treated C2C12 cells in a concentration-dependent manner. DNA damage induced by H2O2 was also markedly attenuated in the presence of CFE, which was associated with suppression of ROS generation. In addition, H2O2 reduced mitochondrial membrane potential and caused downregulation of Bcl-2 and upregulation of Bax expression, although these were abrogated by CFE pretreatment. Moreover, CFE blocked H2O2-induced cytosolic release of cytochrome c, activation of caspase-9 and caspase-3, and degradation of poly (ADP-ribose) polymerase. CONCLUSION: Taken together, the present results demonstrate that CFE could protect C2C12 cells from H2O2-induced damage by eliminating ROS generation, thereby blocking mitochondria-mediated apoptosis pathway. These results indicate that CFE has therapeutic potential for the prevention and treatment of oxidative stress-mediated myoblast injury.

VDR regulates simulated microgravity-induced atrophy in C2C12 myotubes.

Muscle wasting is a major problem leading to reduced quality of life and higher risks of mortality and various diseases. Muscle atrophy is caused by multiple conditions in which protein degradation exceeds its synthesis, including disuse, malnutrition, and microgravity. While Vitamin D receptor (VDR) is well known to regulate calcium and phosphate metabolism to maintain bone, recent studies have shown that VDR also plays roles in skeletal muscle development and homeostasis. Moreover, its expression is upregulated in muscle undergoing atrophy as well as after muscle injury. Here we show that VDR regulates simulated microgravity-induced atrophy in C2C12 myotubes in vitro. After 8 h of microgravity simulated using 3D-clinorotation, the VDR-binding motif was associated with chromatin regions closed by the simulated microgravity and enhancer regions inactivated by it, which suggests VDR mediates repression of enhancers. In addition, VDR was induced and translocated into the nuclei in response to simulated microgravity. VDR-deficient C2C12 myotubes showed resistance to simulated microgravity-induced atrophy and reduced induction of FBXO32, an atrophy-associated ubiquitin ligase. These results demonstrate that VDR contributes to the regulation of simulated microgravity-induced atrophy at least in part by controlling expression of atrophy-related genes.

Dynamics of muscle growth and regeneration: Lessons from the teleost.

The processes of myogenesis during both development and regeneration share a number of similarities across both amniotes and teleosts. In amniotes, the process of muscle formation is considered largely biphasic, with developmental myogenesis occurring through hyperplastic fibre deposition and postnatal muscle growth driven through hypertrophy of existing fibres. In contrast, teleosts continue generating new muscle fibres during adult myogenesis through a process of eternal hyperplasia using a dedicated stem cell system termed the external cell layer. During developmental and regenerative myogenesis alike, muscle progenitors interact with their niche to receive cues guiding their transition into myoblasts and ultimately mature myofibres. During development, muscle precursors receive input from neighbouring embryological tissues; however, during repair, this role is fulfilled by other injury resident cell types, such as those of the innate immune response. Recent work has focused on the role of macrophages as a pro-regenerative cell type which provides input to muscle satellite cells during regenerative myogenesis. As zebrafish harbour a satellite cell system analogous to that of mammals, the processes of regeneration can be interrogated in vivo with the imaging intensive approaches afforded in the zebrafish system. This review discusses the strengths of zebrafish with a focus on both the similarities and differences to amniote myogenesis during both development and repair.

Regenerative strategies for the consequences of myocardial infarction: Chronological indication and upcoming visions.

Heart muscle injury and an elevated troponin level signify myocardial infarction (MI), which may result in defective and uncoordinated segments, reduced cardiac output, and ultimately, death. Physicians apply thrombolytic therapy, coronary artery bypass graft (CABG) surgery, or percutaneous coronary intervention (PCI) to recanalize and restore blood flow to the coronary arteries, albeit they were not convincingly able to solve the heart problems. Thus, researchers aim to introduce novel substitutional therapies for regenerating and functionalizing damaged cardiac tissue based on engineering concepts. Cell-based engineering approaches, utilizing biomaterials, gene, drug, growth factor delivery systems, and tissue engineering are the most leading studies in the field of heart regeneration. Also, understanding the primary cause of MI and thus selecting the most efficient treatment method can be enhanced by preparing microdevices so-called heart-on-a-chip. In this regard, microfluidic approaches can be used as diagnostic platforms or drug screening in cardiac disease treatment. Additionally, bioprinting technique with whole organ 3D printing of human heart with major vessels, cardiomyocytes and endothelial cells can be an ideal goal for cardiac tissue engineering and remarkable achievement in near future. Consequently, this review discusses the different aspects, advancements, and challenges of the mentioned methods with presenting the advantages and disadvantages, chronological indications, and application prospects of various novel therapeutic approaches.

Synthesis and Exon-Skipping Properties of a 3'-Ursodeoxycholic Acid-Conjugated Oligonucleotide Targeting DMD Pre-mRNA: Pre-Synthetic versus Post-Synthetic Approach.

Steric blocking antisense oligonucleotides (ASO) are promising tools for splice modulation such as exon-skipping, although their therapeutic effect may be compromised by insufficient delivery. To address this issue, we investigated the synthesis of a 20-mer 2'-OMe PS oligonucleotide conjugated at 3'-end with ursodeoxycholic acid (UDCA) involved in the targeting of human DMD exon 51, by exploiting both a pre-synthetic and a solution phase approach. The two approaches have been compared. Both strategies successfully provided the desired ASO 51 3'-UDC in good yield and purity. It should be pointed out that the pre-synthetic approach insured better yields and proved to be more cost-effective. The exon skipping efficiency of the conjugated oligonucleotide was evaluated in myogenic cell lines and compared to that of unconjugated one: a better performance was determined for ASO 51 3'-UDC with an average 9.5-fold increase with respect to ASO 51.

Skeletal stem cell fate defects caused by Pdgfrb activating mutation.

Autosomal dominant PDGFRß gain-of-function mutations in mice and humans cause a spectrum of wasting and overgrowth disorders afflicting the skeleton and other connective tissues, but the cellular origin of these disorders remains unknown. We demonstrate that skeletal stem cells (SSCs) isolated from mice with a gain-of-function D849V point mutation in PDGFRß exhibit colony formation defects that parallel the wasting or overgrowth phenotypes of the mice. Single-cell RNA transcriptomics with SSC-derived polyclonal colonies demonstrates alterations in osteogenic and chondrogenic precursors caused by PDGFRßD849V. Mutant cells undergo poor osteogenesis in vitro with increased expression of Sox9 and other chondrogenic markers. Mice with PDGFRßD849V exhibit osteopenia. Increased STAT5 phosphorylation and overexpression of Igf1 and Socs2 in PDGFRßD849V cells suggests that overgrowth in mice involves PDGFRßD849V activating the STAT5-IGF1 axis locally in the skeleton. Our study establishes that PDGFRßD849V causes osteopenic skeletal phenotypes that are associated with intrinsic changes in SSCs, promoting chondrogenesis over osteogenesis.

Molecular Imaging of Human Skeletal Myoblasts (huSKM) in Mouse Post-Infarction Myocardium.

Current treatment protocols for myocardial infarction improve the outcome of disease to some extent but do not provide the clue for full regeneration of the heart tissues. An increasing body of evidence has shown that transplantation of cells may lead to some organ recovery. However, the optimal stem cell population has not been yet identified. We would like to propose a novel pro-regenerative treatment for post-infarction heart based on the combination of human skeletal myoblasts (huSkM) and mesenchymal stem cells (MSCs). huSkM native or overexpressing gene coding for Cx43 (huSKMCx43) alone or combined with MSCs were delivered in four cellular therapeutic variants into the healthy and post-infarction heart of mice while using molecular reporter probes. Single-Photon Emission Computed Tomography/Computed Tomography (SPECT/CT) performed right after cell delivery and 24 h later revealed a trend towards an increase in the isotopic uptake in the post-infarction group of animals treated by a combination of huSkMCx43 with MSC. Bioluminescent imaging (BLI) showed the highest increase in firefly luciferase (fluc) signal intensity in post-infarction heart treated with combination of huSkM and MSCs vs. huSkM alone (p < 0.0001). In healthy myocardium, however, nanoluciferase signal (nanoluc) intensity varied markedly between animals treated with stem cell populations either alone or in combinations with the tendency to be simply decreased. Therefore, our observations seem to show that MSCs supported viability, engraftment, and even proliferation of huSkM in the post-infarction heart.

Skeletal muscle bioenergetic health and function in people living with HIV: association with glucose tolerance and alcohol use.

At-risk alcohol use is prevalent and increases dysglycemia among people living with human immunodeficiency virus (PLWH). Skeletal muscle (SKM) bioenergetic dysregulation is implicated in dysglycemia and type 2 diabetes. The objective of this study was to determine the relationship between at-risk alcohol, glucose tolerance, and SKM bioenergetic function in PLWH. Thirty-five PLWH (11 females, 24 males, age: 53 ± 9 yr, body mass index: 29.0 ± 6.6 kg/m2) with elevated fasting glucose enrolled in the ALIVE-Ex study provided medical history and alcohol use information [Alcohol Use Disorders Identification Test (AUDIT)], then underwent an oral glucose tolerance test (OGTT) and SKM biopsy. Bioenergetic health and function and mitochondrial volume were measured in isolated myoblasts. Mitochondrial gene expression was measured in SKM. Linear regression adjusting for age, sex, and smoking was performed to examine the relationship between glucose tolerance (2-h glucose post-OGTT), AUDIT, and their interaction with each outcome measure. Negative indicators of bioenergetic health were significantly (P < 0.05) greater with higher 2-h glucose (proton leak) and AUDIT (proton leak, nonmitochondrial oxygen consumption, and bioenergetic health index). Mitochondrial volume was increased with the interaction of higher 2-h glucose and AUDIT. Mitochondrial gene expression decreased with higher 2-h glucose (TFAM, PGC1B, PPARG, MFN1), AUDIT (MFN1, DRP1, MFF), and their interaction (PPARG, PPARD, MFF). Decreased expression of mitochondrial genes were coupled with increased mitochondrial volume and decreased bioenergetic health in SKM of PLWH with higher AUDIT and 2-h glucose. We hypothesize these mechanisms reflect poorer mitochondrial health and may precede overt SKM bioenergetic dysregulation observed in type 2 diabetes.

Neddylation Regulates Class IIa and III Histone Deacetylases to Mediate Myoblast Differentiation.

As the largest tissue in the body, skeletal muscle has multiple functions in movement and energy metabolism. Skeletal myogenesis is controlled by a transcriptional cascade including a set of muscle regulatory factors (MRFs) that includes Myogenic Differentiation 1 (MYOD1), Myocyte Enhancer Factor 2 (MEF2), and Myogenin (MYOG), which direct the fusion of myogenic myoblasts into multinucleated myotubes. Neddylation is a posttranslational modification that covalently conjugates ubiquitin-like NEDD8 (neural precursor cell expressed, developmentally downregulated 8) to protein targets. Inhibition of neddylation impairs muscle differentiation; however, the underlying molecular mechanisms remain less explored. Here, we report that neddylation is temporally regulated during myoblast differentiation. Inhibition of neddylation through pharmacological blockade using MLN4924 (Pevonedistat) or genetic deletion of NEDD8 Activating Enzyme E1 Subunit 1 (NAE1), a subunit of the E1 neddylation-activating enzyme, blocks terminal myoblast differentiation partially through repressing MYOG expression. Mechanistically, we found that neddylation deficiency enhances the mRNA and protein expressions of class IIa histone deacetylases 4 and 5 (HDAC4 and 5) and prevents the downregulation and nuclear export of class III HDAC (NAD-Dependent Protein Deacetylase Sirtuin-1, SIRT1), all of which have been shown to repress MYOD1-mediated MYOG transcriptional activation. Together, our findings for the first time identify the crucial role of neddylation in mediating class IIa and III HDAC co-repressors to control myogenic program and provide new insights into the mechanisms of muscle disease and regeneration.

Filopodia powered by class x myosin promote fusion of mammalian myoblasts.

Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.

Simulated microgravity accelerates aging of human skeletal muscle myoblasts at the single cell level.

Earth's gravity is essential for maintaining skeletal muscle mass and function in the body. The role of gravity in the myogenic mechanism has been studied with animal experiments in the International Space Station. Recently, gravity-control devices allow to study the effects of gravity on cultured cells on the ground. This study demonstrated that simulated microgravity accelerated aging of human skeletal muscle myoblasts in an in-vitro culture. The microgravity culture induced a significant decrease in cell proliferation and an enlargement of the cytoskeleton and nucleus of cells. Similar changes are often observed in aged myoblasts following several passages. In fact, by the microgravity culture the expression of senescence associated ß-Gal was significantly enhanced, and some muscle-specific proteins decreased in the enlarged cells. Importantly, these microgravity effects remained with the cells even after a return to normal gravity conditions. Consequently, the microgravity-affected myoblasts demonstrated a reduced capability of differentiation into myotubes. In the body, it is difficult to interpret the disability of microgravity-affected myoblasts, since muscle regeneration is linked to the supply of new myogenic cells. Therefore, our in-vitro cell culture study will be advantageous to better understand the role of each type of myogenic cell in human muscle without gravitational stress at the single cell level.

Pathological Mechanism of a Constitutively Active Form of Stromal Interaction Molecule 1 in Skeletal Muscle.

Stromal interaction molecule 1 (STIM1) is the main protein that, along with Orai1, mediates store-operated Ca2+ entry (SOCE) in skeletal muscle. Abnormal SOCE due to mutations in STIM1 is one of the causes of human skeletal muscle diseases. STIM1-R304Q (a constitutively active form of STIM1) has been found in human patients with skeletal muscle phenotypes such as muscle weakness, myalgia, muscle stiffness, and contracture. However, the pathological mechanism(s) of STIM1-R304Q in skeletal muscle have not been well studied. To examine the pathological mechanism(s) of STIM1-R304Q in skeletal muscle, STIM1-R304Q was expressed in mouse primary skeletal myotubes, and the properties of the skeletal myotubes were examined using single-myotube Ca2+ imaging, transmission electron microscopy (TEM), and biochemical approaches. STIM1-R304Q did not interfere with the terminal differentiation of skeletal myoblasts to myotubes and retained the ability of STIM1 to attenuate dihydropyridine receptor (DHPR) activity. STIM1-R304Q induced hyper-SOCE (that exceeded the SOCE by wild-type STIM1) by affecting both the amplitude and the onset rate of SOCE. Unlike that by wild-type STIM1, hyper-SOCE by STIM1-R304Q contributed to a disturbance in Ca2+ distribution between the cytosol and the sarcoplasmic reticulum (SR) (high Ca2+ in the cytosol and low Ca2+ in the SR). Moreover, the hyper-SOCE and the high cytosolic Ca2+ level induced by STIM1-R304Q involve changes in mitochondrial shape. Therefore, a series of these cellular defects induced by STIM1-R304Q could induce deleterious skeletal muscle phenotypes in human patients carrying STIM1-R304Q.

Dexamethasone Inhibits the Pro-Angiogenic Potential of Primary Human Myoblasts.

Tissue regeneration depends on the complex processes of angiogenesis, inflammation and wound healing. Regarding muscle tissue, glucocorticoids (GCs) inhibit pro-inflammatory signalling and angiogenesis and lead to muscle atrophy. Our hypothesis is that the synthetic GC dexamethasone (dex) impairs angiogenesis leading to muscle atrophy or inhibited muscle regeneration. Therefore, this study aims to elucidate the effect of dexamethasone on HUVECs under different conditions in mono- and co-culture with myoblasts to evaluate growth behavior and dex impact with regard to muscle atrophy and muscle regeneration. Viability assays, qPCR, immunofluorescence as well as ELISAs were performed on HUVECs, and human primary myoblasts seeded under different culture conditions. Our results show that dex had a higher impact on the tube formation when HUVECs were maintained with VEGF. Gene expression was not influenced by dex and was independent of cells growing in a 2D or 3D matrix. In co-culture CD31 expression was suppressed after incubation with dex and gene expression analysis revealed that dex enhanced expression of myogenic transcription factors, but repressed angiogenic factors. Moreover, dex inhibited the VEGF mediated pro angiogenic effect of myoblasts and inhibited expression of angiogenic inducers in the co-culture model. This is the first study describing a co-culture of human primary myoblast and HUVECs maintained under different conditions. Our results indicate that dex affects angiogenesis via inhibition of VEGF release at least in myoblasts, which could be responsible not only for the development of muscle atrophy after dex administration, but also for inhibition of muscle regeneration after vascular damage.