Differentiation Of Muscle Precursor Cells Research Articles

Myogenetic Oligodeoxynucleotide (myoDN) Recovers the Differentiation of Skeletal Muscle Myoblasts Deteriorated by Diabetes Mellitus.

Skeletal muscle wasting in patients with diabetes mellitus (DM) is a complication of decreased muscle mass and strength, and is a serious risk factor that may result in mortality. Deteriorated differentiation of muscle precursor cells, called myoblasts, in DM patients is considered to be one of the causes of muscle wasting. We recently developed myogenetic oligodeoxynucleotides (myoDNs), which are 18-base single-strand DNAs that promote myoblast differentiation by targeting nucleolin. Herein, we report the applicability of a myoDN, iSN04, to myoblasts isolated from patients with type 1 and type 2 DM. Myogenesis of DM myoblasts was exacerbated concordantly with a delayed shift of myogenic transcription and induction of interleukins. Analogous phenotypes were reproduced in healthy myoblasts cultured with excessive glucose or palmitic acid, mimicking hyperglycemia or hyperlipidemia. iSN04 treatment recovered the deteriorated differentiation of plural DM myoblasts by downregulating myostatin and interleukin-8 (IL-8). iSN04 also ameliorated the impaired myogenic differentiation induced by glucose or palmitic acid. These results demonstrate that myoDNs can directly facilitate myoblast differentiation in DM patients, making them novel candidates for nucleic acid drugs to treat muscle wasting in patients with DM.

Divergent effects of myogenic differentiation and diabetes on the capacity for muscle precursor cell adipogenic differentiation in a fibrin matrix

The development of ectopic adipose tissue in skeletal muscle is associated with several skeletal muscle and metabolic pathologies, including Type II Diabetes Mellitus. The adipogenic differentiation of muscle precursor cells (MPCs) has been postulated to occur in skeletal muscle in vivo in a three-dimensional (3-D) configuration; therefore, it is appropriate to investigate this phenomenon using 3-D matrices in vitro. The capacity for MPC adipogenic differentiation in a 3-D environment was investigated in fibrin hydrogels by treating MPCs derived from healthy or diabetic animals with adipogenic induction medias that differed in their ability to increase lipid accumulation and activate the expression of genes associated with adipogenic differentiation (peroxisome proliferator-activated receptor gamma (PPARG), adiponectin (ADIPOQ), and fatty acid synthase (FAS)). The capacity for adipogenic differentiation was diminished, but not prevented, if myogenic differentiation preceded MPC exposure to adipogenic induction conditions. Conversely, adipogenic differentiation was greater in hydrogels containing MPCs from diabetic rats as compared to those derived from lean rats, as evidenced by an increase in lipid accumulation and adipogenic gene expression. Collectively, the data herein support a role for the MPCs in adipogenesis in a 3-D environment and that they may contribute to the ectopic accumulation of adipose tissue. The observation that the potential for adipogenic differentiation is maintained even after a period of myogenic differentiation alludes to the possibility that adipogenesis may occur during different phases of muscle development. Finally, the increase in adipogenic differentiation in hydrogels containing MPCs derived from diabetic animals provides strong evidence that a pathological environment in vivo increases their capacity for adipogenesis.

Biofabrication of tissue-specific extracellular matrix proteins to enhance the expansion and differentiation of skeletal muscle progenitor cells

Skeletal muscle precursor cells (MPCs) are considered key candidates for cell therapy in the treatment of skeletal muscle dysfunction due to injury, disease, or aging. However, expansion of a sufficient number of functional skeletal muscle cells in vitro from a small tissue biopsy has been challenging due to changes in the phenotypic expression of these cells under nonnatural microenvironmental or traditional culture conditions. This review provides an overview of recent progress in the design and biofabrication of advanced tissue-specific extracellular matrix (ECM) proteins for use in the enhancement of expansion and differentiation of MPCs for cell therapy and 3D bioprinting. We start with a brief introduction about the existing progress, drawbacks, and emerging challenges in the culture and maintenance of long term primary human MPCs for cell therapy. With regard to MPC proliferation, elongation, fusion, and differentiation into mature myofibers, we systematically summarize the benefits and limitations of recent progress. The importance of tissue-specific ECM in skeletal muscle regeneration is discussed, in particular, the mechanisms, rationale, strategy, and methodologies for using tissue-specific ECM proteins for myogenesis in 2D and 3D culture environments. Furthermore, perspectives on the challenges in developing tissue-specific ECM proteins for cell therapy using human MPCs are described. Finally, we propose potential strategies for overcoming the challenges in the development of advanced tissue-specific ECM proteins for promoting cell therapy with human skeletal muscle cells.

Ascorbic acid diminishes bone morphogenetic protein 2-induced osteogenic differentiation of muscle precursor cells.

Muscle precursor cells (MPC) are integral to the maintenance of skeletal muscle and have recently been implicated in playing a role in bone repair. The primary objective of this study was to understand better the role of oxidative stress during the osteogenic differentiation of MPCs. Muscle precursor cells were treated with various combinations of ascorbic acid (AA), bone morphogenetic protein (BMP)-2, and either a superoxide dismutase analog (4-hydroxy-TEMPO [TEMPOL]) or polyethyleneglycol-conjugated catalase. Muscle precursor cell proliferation and differentiation were determined, and alkaline phosphatase activity was measured as an index of osteogenic differentiation. After treatment with 200 μM AA, superoxide was increased 1.5-fold, whereas AA in combination with 100 ng/ml BMP-2 did not increase alkaline phosphatase (ALP) activity. When cells were treated with TEMPOL in combination with 100 ng/ml BMP-2 and 200 μM AA, ALP activity significantly increased. These data suggest that increasing oxidative stress with AA induces sublethal oxidative stress that prevents BMP-2-induced osteogenic differentiation of MPCs. Muscle Nerve 59:501-508, 2019.

Kinin-B2 Receptor Activity in Skeletal Muscle Regeneration and Myoblast Differentiation.

The bioactive peptide bradykinin obtained from cleavage of precursor kininogens activates the kinin-B2 receptor functioning in induction of inflammation and vasodilatation. In addition, bradykinin participates in kidney and cardiovascular development and neuronal and muscle differentiation. Here we show that kinin-B2 receptors are expressed throughout differentiation of murine C2C12 myoblasts into myotubes. An autocrine loop between receptor activation and bradykinin secretion is suggested, since bradykinin secretion is significantly reduced in the presence of the kinin-B2 receptor antagonist HOE-140 during differentiation. Expression of skeletal muscle markers and regenerative capacity were decreased after pharmacological inhibition or genetic ablation of the B2 receptor, while its antagonism increased the number of myoblasts in culture. In summary, the present work reveals to date no functions described for the B2 receptor in muscle regeneration due to the control of proliferation and differentiation of muscle precursor cells.

Abstract B22: Uncovering the role of lysyl oxidase-like 2 in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common soft-tissue malignancy in childhood and adolescence that originates as a consequence of regulatory disruption in the growth and differentiation of muscle precursor cells. Members of the lysyl oxidase family of proteins (LOX and LOXL1-4) are amine oxidases that catalyze the covalent crosslinking of collagen and elastin in the extracellular matrix. Apart from their traditional role, novel functions have been attributed to these proteins related to tumor progression. In this study we sought to unravel the potential role of this family of proteins during the progression of RMS. By qRT-PCR and Western blot, lysyl oxidase-like 2 (LOXL2) was found overexpressed in RMS cell lines compared to other sarcoma cells. Then we characterized LOXL2 in RMS cell lines by analyzing the subcellular localization by immunofluorescence and subcellular fractionation. Post-translational modifications were also analyzed: glycosylation and proteolytic processing by tunicamycin treatment and secretion by analysis of the conditioned media by Western blot. Moreover, we characterized the neoplastic phenotype after silencing LOXL2 in two RMS cell lines (RH4 and CW9019). Stable LOXL2 knockdown resulted in a decrease in their clonogenic, proliferative, and cell migratory capabilities. On the other hand, reexpression of LOXL2 in RH28 cells using wild-type or mutated (catalytically inactive) constructs resulted in an increase in cell proliferation and migration, independently of its catalytic activity. Finally, in vivo experiments using an orthotopic model of tumor generation inside the muscle will be carried out to further evaluate the tumorigenic role of LOXL2. Taking all into consideration, our results suggest an oncogenic role of LOXL2 in RMS. Citation Format: Olga Almacellas Rabaiget, Juan Huertas Martínez, Silvia Garcia Monclús, Santiago Rello Varona, David Herrero Martín, Roser López Alemany, Oscar M. Tirado. Uncovering the role of lysyl oxidase-like 2 in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B22.

* Tissue-Specific Extracellular Matrix Enhances Skeletal Muscle Precursor Cell Expansion and Differentiation for Potential Application in Cell Therapy.

Skeletal muscle precursor cells (MPCs) are considered a key candidate for cell therapy in the treatment of skeletal muscle dysfunction due to injury, disease, or age. However, expansion of a sufficient number of functional skeletal muscle cells in vitro from a small tissue biopsy has been challenging due to changes in phenotypic expression of these cells under traditional culture conditions. Thus, the aim of the study was to develop a better culture system for the expansion and myo-differentiation of MPCs that could further be used for therapy. For this purpose, we developed an ideal method of tissue decellularization and compared the ability of different matrices to support MPC growth and differentiation. Porcine-derived skeletal muscle and liver and kidney extracellular matrix (ECM) were generated by decellularization methods consisting of distilled water, 0.2 mg/mL DNase, or 5% fetal bovine serum. Acellular matrices were further homogenized, dissolved, and combined with a hyaluronic acid-based hydrogel decorated with heparin (ECM-HA-HP). The cell proliferation and myogenic differentiation capacity of human MPCs were assessed when grown on gel alone, ECM, or each ECM-HA-HP substrate. Human MPC proliferation was significantly enhanced when cultured on the ECM-HA-HP substrates compared to the other substrates tested, with the greatest proliferation on the muscle ECM-HA-HP (mECM-HA-HP) substrate. The number of differentiated myotubes was significantly increased on the mECM-HA-HP substrate compared to the other gel-ECM substrates, as well as the numbers of MPCs expressing specific myogenic cell markers (i.e., myosin, desmin, myoD, and myf5). In conclusion, skeletal mECM-HA-HP as a culture substrate provided an optimal culture microenvironment potentially due to its similarity to the in vivo environment. These data suggest a potential use of skeletal muscle-derived ECM gel for the expansion and differentiation of human MPCs for cell-based therapy for skeletal muscle dysfunction.

BAG3 (Bcl-2-Associated Athanogene-3) Coding Variant in Mice Determines Susceptibility to Ischemic Limb Muscle Myopathy by Directing Autophagy.

Critical limb ischemia is a manifestation of peripheral artery disease that carries significant mortality and morbidity risk in humans, although its genetic determinants remain largely unknown. We previously discovered 2 overlapping quantitative trait loci in mice, Lsq-1 and Civq-1, that affected limb muscle survival and stroke volume after femoral artery or middle cerebral artery ligation, respectively. Here, we report that a Bag3 variant (Ile81Met) segregates with tissue protection from hind-limb ischemia. We treated mice with either adeno-associated viruses encoding a control (green fluorescent protein) or 2 BAG3 (Bcl-2-associated athanogene-3) variants, namely Met81 or Ile81, and subjected the mice to hind-limb ischemia. We found that the BAG3 Ile81Met variant in the C57BL/6 (BL6) mouse background segregates with protection from tissue necrosis in a shorter congenic fragment of Lsq-1 (C.B6-Lsq1-3). BALB/c mice treated with adeno-associated virus encoding the BL6 BAG3 variant (Ile81; n=25) displayed reduced limb-tissue necrosis and increased limb tissue perfusion compared with Met81- (n=25) or green fluorescent protein- (n=29) expressing animals. BAG3Ile81, but not BAG3Met81, improved ischemic muscle myopathy and muscle precursor cell differentiation and improved muscle regeneration in a separate, toxin-induced model of injury. Systemic injection of adeno-associated virus-BAG3Ile81 (n=9), but not BAG3Met81 (n=10) or green fluorescent protein (n=5), improved ischemic limb blood flow and limb muscle histology and restored muscle function (force production). Compared with BAG3Met81, BAG3Ile81 displayed improved binding to the small heat shock protein (HspB8) in ischemic skeletal muscle cells and enhanced ischemic muscle autophagic flux. Taken together, our data demonstrate that genetic variation in BAG3 plays an important role in the prevention of ischemic tissue necrosis. These results highlight a pathway that preserves tissue survival and muscle function in the setting of ischemia.

Magnetization Transfer MR Imaging to Monitor Muscle Tissue Formation during Myogenic in Vivo Differentiation of Muscle Precursor Cells.

Purpose To determine whether magnetization transfer (MT) magnetic resonance (MR) imaging may serve as a quantitative measure of the degree of fiber formation during differentiation of muscle precursor cells into engineered muscle tissue as a potential noninvasive monitoring tool in mice. Materials and Methods The study was approved by the local ethics committee (no. StV 01/2008) and the local Veterinary Office (license no. 99/2013). Human muscle progenitor cells (hMPCs) derived from rectus abdominis muscles were subcutaneously injected into CD-1 nude mice (CD-1 nude mice, Crl:CD1-Foxn1nu; Charles River Laboratories, Wilmington, Mass) for development of muscle tissue. The mice underwent MR imaging examinations at 4.7 T at days 1, 3, 7, 14, 21, and 28 after cell transplantation by using a gradient-echo sequence with an MT prepulse and systematic variation of the off-resonance frequency (50-37 500 Hz) at an amplitude of 800°. Direct saturation was estimated from a Bloch equation simulation. The MT ratio (MTR) was correlated to immunohistochemistry findings, Western blot results, and results of myography. Data were analyzed by using one-way or two-way analysis of variance with the Sidak or Tukey multiple comparisons test. Results In the reference skeletal muscle, highest MT was found for 2500 Hz off-resonance frequency with an MTR ± standard deviation of 57.5% ± 3.5. The developing muscle tissue exhibited increasing MT values during the 28 days of myogenic in vivo differentiation and did not reach the values of native skeletal muscle. Mean values of MTR (2500 Hz) for hMPCs were 27.6% ± 6.3 (day 1), 24.7% ± 8.7 (day 3), 28.2% ± 5.7 (day 7), 35.9% ± 5.0 (day 14), 37.0% ± 7.9 (day 21), and 39.9% ± 8.1 (day 28). The results from MT MR imaging correlated qualitatively well with muscle tissue expression of specific skeletal markers, as well as muscle contractility. Conclusion MT MR imaging may be used to noninvasively monitor the process of myogenic in vivo differentiation of hMPCs as a biomarker of the quantity and quality of muscle fiber formation. © RSNA, 2016 Online supplemental material is available for this article.

Uncovering the role of lysyl oxidase-like 2 (LOXL2) in rhabdomyosarcoma

Introduction: Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in childhood and adolescence that originates as a consequence of regulatory disruption in the growth and differentiation of muscle precursor cells. Previous results of our group deciphered the epigenetic profiling of RMS, compared to healthy tissue, revealing the hypomethylation of a potential enhancer of LOXL2. LOXL2 is an amine oxidase that catalyzes the covalent crosslinking of collagen and elastin in the extracellular matrix. Apart from its traditional role, novel functions have been attributed to LOXL2 related to tumor progression. Methods: Infinium 450k Illumina methylation array of RMS cell lines and patients. Western blot, qRT-PCR, clonogenic, proliferation and migration assays. Results: We first confirmed the overexpression of LOXL2 in RMS cell lines compared to other sarcoma cells by qRT-PCR and Western blot. Moreover, we characterized the neoplastic phenotype after silencing LOXL2 expression in RH4 cell line. LOXL2 silencing resulted in a decrease in the clonogenic capacity, proliferation and cell migration. On the other hand, gain of function experiments in LOXL2 under-expressed cells (RH28) using wild type or mutated (catalytically inactive) constructs of LOXL2 will be crucial to further explore its role in RMS. Conclusion: Our preliminary results suggest an oncogenic role of LOXL2 in RMS.

Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs.

Although there are many studies focusing on the molecular pathways underlying lung vascular morphogenesis, the extracellular matrix (ECM)-dependent regulation of mesenchymal cell differentiation in vascular smooth muscle development needs better understanding. In this study, we demonstrate that the paired related homeobox gene transcription factor Prx1 maintains the elastic ECM properties, which are essential for vascular smooth muscle precursor cell differentiation. We have found that Prx1(null) mouse lungs exhibit defective vascular smooth muscle development, downregulated elastic ECM expression, and compromised transforming growth factor (TGF)-β localization and signaling. Further characterization of ECM properties using decellularized lung ECM scaffolds derived from Prx1 mice demonstrated that Prx1 is required to maintain lung ECM stiffness. The results of cell culture using stiffness-controlled 2-D and 3-D synthetic substrates confirmed that Prx1-dependent ECM stiffness is essential for promotion of smooth muscle precursor differentiation for effective TGF-β stimulation. Supporting these results, both decellularized Prx1(null) lung ECM and Prx1(WT) (wild type) ECM scaffolds with blocked TGF-β failed to support mesenchymal cell to 3-D smooth muscle cell differentiation. These results suggest a novel ECM-dependent regulatory pathway of lung vascular development wherein Prx1 regulates lung vascular smooth muscle precursor development by coordinating the ECM biophysical and biochemical properties.

Electrical stimulation by enzymatic biofuel cell to promote proliferation, migration and differentiation of muscle precursor cells

Electrical stimulation is a very important biophysical cue for skeletal muscle maintenance and myotube formation. The absence of electrical signals from motor neurons causes denervated muscles to atrophy. Herein, we investigate for the first time the utility of an enzymatic biofuel cell (EBFC) as a promising means for mimicking native electrical stimulation. EBFC was set up using two different enzymes: one was glucose oxidase (GOX) used for the generation of anodic current followed by the oxidation of glucose; the other was Bilirubin oxidase (BOD) for the generation of cathodic current followed by the reduction of oxygen. We studied the behaviors of muscle precursor cells (MPCs) in terms of proliferation, migration and differentiation under different electrical conditions. The EBFC electrical stimulations significantly increased cell proliferation and migration. Furthermore, the electrical stimulations promoted the differentiation of cells into myotube formation based on expressions at the gene and protein levels. The EBFC set up, with its free forms adjustable to any implant design, was subsequently applied to the nanofiber scaffolding system. The MPCs were demonstrated to be stimulated in a similar manner as the 2D culture conditions, suggesting potential applications of the EBFC system for muscle repair and regeneration.

MR imaging relaxometry allows noninvasive characterization of in vivo differentiation of muscle precursor cells.

To demonstrate the feasibility of in vivo monitoring of the myogenic differentiation process from human muscle precursor cells to mature skeletal muscle tissue by measuring characteristic magnetic resonance (MR) imaging relaxation and diffusion properties as a potential noninvasive diagnostic tool in muscle cell therapy. The study was approved by the ethics committee for studies in humans and the animal care committee. The hypothesis was tested by means of subcutaneous injection of human muscle precursor cells from the rectus abdominis muscle into nude mice (n = 18). Animals injected with human fibroblasts, prostate cancer cells, or collagen served as control animals (four in each group). T1, T2, T2*, and apparent diffusion coefficients (ADCs) were measured at 4.7-T MR imaging. MR imaging parameters were statistically evaluated by using analysis of variance with Bonferroni correction. The engineered muscle was characterized by means of immunofluorescence, Western blot, and contraction assays. Muscle tissue in the early stages of the differentiation process exhibited distinctly higher T1 (mean ± standard deviation, 2242 msec ± 116), T2 (224 msec ± 18), and T2* (33.3 msec ± 3.6) values and ADCs (1.53 × 10(-3) mm(2)/sec ± 0.03) compared with those of skeletal muscle. The muscle precursor cells exhibited a nonspecific pattern compared with that in control animals in the early stages. During differentiation, the relaxation and diffusion parameters decreased and approached the values for mature skeletal muscle tissue: T1, 1386 msec ± 88; T2, 32.0 msec ± 4.3; T2*, 10.8 msec ± 0.8; ADC, 1.39 × 10(-3) mm(2)/sec ± 0.02 (reference erector spinae muscle tissue: T1, 1417 msec ± 106; T2, 31.0 msec ± 2.4; T2*, 11.3 msec ± 1.7; and ADC, 1.40 × 10(-3) mm(2)/sec ± 0.03). MR imaging relaxation and diffusion measurements can be used as potential biomarkers for noninvasive in vivo monitoring of the myogenic differentiation process from muscle precursor cells to mature skeletal muscle tissue in muscle cell therapy.

Sharp-1 regulates TGF-β signaling and skeletal muscle regeneration

Sharp-1 is a basic helix-loop-helix (bHLH) transcriptional repressor that is involved in a number of cellular processes. Our previous studies have demonstrated that Sharp-1 is a negative regulator of skeletal myogenesis and it blocks differentiation of muscle precursor cells by modulating the activity of MyoD. In order to understand its role in pre- and post-natal myogenesis, we assessed skeletal muscle development and freeze-injury-induced regeneration in Sharp-1-deficient mice. We show that embryonic skeletal muscle development is not impaired in the absence of Sharp-1; however, post-natally, the regenerative capacity is compromised. Although the initial phases of injury-induced regeneration proceed normally in Sharp-1(-/-) mice, during late stages, the mutant muscle exhibits necrotic fibers, calcium deposits and fibrosis. TGF-β expression, as well as levels of phosphorylated Smad2 and Smad3, are sustained in the mutant tissue and treatment with decorin, which blocks TGF-β signaling, improves the histopathology of Sharp-1(-/-) injured muscles. In vitro, Sharp-1 associates with Smad3, and its overexpression inhibits TGF-β- and Smad3-mediated expression of extracellular matrix genes in myofibroblasts. These results demonstrate that Sharp-1 regulates muscle regenerative capacity, at least in part, by modulation of TGF-β signaling.

P.1.19 Impaired viability of muscle precursor cells in muscular dystrophy with glycosylation defects and amelioration of its severe phenotype by limited gene expression

A group of muscular dystrophies, dystroglycanopathy, is caused by abnormalities in post-translational modifications of dystroglycan (DG). To better understand the pathophysiological roles of DG modification and to establish effective treatment for dystroglycanopathy, we generated 2 distinct conditional knock-out (cKO) mice for fukutin, the first dystroglycanopathy gene identified for Fukuyama congenital muscular dystrophy. The first dystroglycanopathy model – myofiber-selective fukutin-cKO (MCK-fukutin-cKO) mice – showed mild muscular dystrophy. Forced exercise experiments in presymptomatic MCK-fukutin-cKO mice revealed that myofiber membrane fragility triggered disease manifestation. The second dystroglycanopathy model – muscle precursor cell (MPC)-selective cKO (Myf5-fukutin-cKO) mice – exhibited more severe phenotypes of muscular dystrophy. Using an isolated MPC culture system, we demonstrated that defects in the fukutin-dependent modification of DG lead to impairment of MPC proliferation, differentiation, and muscle regeneration. These results suggest that impaired MPC viability contributes to the pathology of dystroglycanopathy. Since our data suggested that frequent cycles of myofiber degeneration/regeneration accelerate substantial and/or functional loss of MPC, we expected that protection from disease-triggering myofiber degeneration provides therapeutic effects even in mouse models with MPC defects; therefore, we restored fukutin expression in myofibers. Adeno-associated virus (AAV)-mediated rescue of fukutin expression that was limited in myofibers successfully ameliorated the severe pathology even after disease progression. In addition, compared to other gene therapy studies, considerably low AAV titers were associated with therapeutic effects. Our findings indicated that fukutin-deficient dystroglycanopathy is a regeneration-defective disorder, and gene therapy is a feasible treatment for the wide range of dystroglycanopathy even after disease progression.

Epigenetic control of type 2 and 3 deiodinases in myogenesis: role of Lysine-specific Demethylase enzyme and FoxO3

The proliferation and differentiation of muscle precursor cells require myogenic regulatory factors and chromatin modifiers whose concerted action dynamically regulates access to DNA and allows reprogramming of cells towards terminal differentiation. Type 2 deiodinase (D2), the thyroid hormone (TH)-activating enzyme, is sharply upregulated during myoblast differentiation, whereas type 3 deiodinase (D3), the TH-inactivating enzyme, is downregulated. The molecular determinants controlling synchronized D2 and D3 expression in muscle differentiation are completely unknown. Here, we report that the histone H3 demethylating enzyme (LSD-1) is essential for transcriptional induction of D2 and repression of D3. LSD-1 relieves the repressive marks (H3-K9me2-3) on the Dio2 promoter and the activation marks (H3-K4me2-3) on the Dio3 promoter. LSD-1 silencing impairs the D2 surge in skeletal muscle differentiation while inducing D3 expression thereby leading to a global decrease in intracellular TH production. Furthermore, endogenous LSD-1 interacts with FoxO3a, and abrogation of FoxO3-DNA binding compromises the ability of LSD-1 to induce D2. Our data reveal a novel epigenetic control of reciprocal deiodinases expression and provide a molecular mechanism by which LSD-1, through the opposite regulation of D2 and D3 expression, acts as a molecular switch that dynamically finely tunes the cellular needs of active TH during myogenesis.

NF‐KB activity functions in primary pericytes in a cell‐ and non‐cell‐autonomous manner to affect myotube formation

Skeletal muscle regeneration following damage relies on proliferation and differentiation of muscle precursor cells (MPCs). We recently observed increased NF-kB activity in vascular-associated muscle resident pericytes following muscle damage in humans. We determined how altered NF-kB activity in human primary pericytes (HPPs) affects their myogenic differentiation (cell-autonomous effects), as well as proliferation and differentiation of co-cultured MPCs (non-cell-autonomous effects). HPPs were transfected with vectors that increased or decreased NF-kB activity. Transfected HPPs were co-cultured with C2 C12 myoblasts under differentiation conditions, and HPP fusion to myotubes was measured. We also co-cultured HPPs with C2 C12 myoblasts and measured proliferation and myotube formation. Inhibition of NF-kB activity increased HPP fusion to C2 C12 myotubes. Moreover, enhanced NF-kB activity in HPPs suppressed differentiation and enhanced proliferation of co-cultured myoblasts. NF-kB activity acts cell-autonomously to inhibit HPP myogenic differentiation and non-cell-autonomously to promote MPC proliferation and suppress MPC differentiation in vitro.

Sharp-1 regulates TGF-β signaling and skeletal muscle regeneration

Sharp-1 is a basic helix-loop-helix (bHLH) transcriptional repressor that is involved in a number of cellular processes. Our previous studies have demonstrated that Sharp-1 is a negative regulator of skeletal myogenesis and it blocks differentiation of muscle precursor cells by modulating the activity of MyoD. In order to understand its role in pre- and post-natal myogenesis, we assessed skeletal muscle development and freeze-injury-induced regeneration in Sharp-1-deficient mice. We show that embryonic skeletal muscle development is not impaired in the absence of Sharp-1; however, post-natally, the regenerative capacity is compromised. Although the initial phases of injury-induced regeneration proceed normally in Sharp-1(-/-) mice, during late stages, the mutant muscle exhibits necrotic fibers, calcium deposits and fibrosis. TGF-β expression, as well as levels of phosphorylated Smad2 and Smad3, are sustained in the mutant tissue and treatment with decorin, which blocks TGF-β signaling, improves the histopathology of Sharp-1(-/-) injured muscles. In vitro, Sharp-1 associates with Smad3, and its overexpression inhibits TGF-β- and Smad3-mediated expression of extracellular matrix genes in myofibroblasts. These results demonstrate that Sharp-1 regulates muscle regenerative capacity, at least in part, by modulation of TGF-β signaling.

Non‐passaged muscle precursor cells from 32‐month old rat skeletal muscle have delayed proliferation and differentiation

The systemic environment and satellite cell dysfunction have been proposed as important contributors in the development of sarcopenia and impaired skeletal muscle regrowth with ageing. In the present study, we investigated effects of serum age on proliferation of muscle precursor cells (MPCs) isolated from skeletal muscles of young and old rats. We examined proliferation and subsequent differentiation of non-passaged MPCs isolated from skeletal muscles of 1-, 3- and 32-month old rats over a 72-h time course, using a serum cross-over design. We found no effect of serum age on MPC proliferation, but we did discover that MPCs isolated from skeletal muscle of 32-month old rats had delayed onset of, and exit from proliferation, compared to MPCs isolated from skeletal muscle of 1-month old rats. Delayed proliferation of MPCs from 32-month old rats was associated with delayed p38 MAPK phosphorylation, and MyoD and p21(Cip1) protein expression. We also demonstrate that MPCs from 32-month old rats exhibited lower levels of muscle creatine kinase mRNA compared to 1-month old rats, but elevated levels of myogenin mRNA, when stimulated to differentiate after 36 h proliferation. These findings suggest that delayed entry and exit of the cell cycle observed in MPCs from 32-month old rats may compromise their ability to respond to differentiation stimuli and subsequently impair myogenic potential of 32-month old skeletal muscle, in this model.

TWEAK in Inclusion-Body Myositis Muscle: Possible Pathogenic Role of a Cytokine Inhibiting Myogenesis

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 exert pleiotropic effects, including regulation of myogenesis. Sporadic inclusion-body myositis (IBM) is the most common muscle disease of the elderly population and leads to severe disability. IBM mesoangioblasts, different from mesoangioblasts in other inflammatory myopathies, display a myogenic differentiation defect. The objective of the present study was to investigate TWEAK-Fn14 expression in IBM and other inflammatory myopathies and explore whether TWEAK modulation affects myogenesis in IBM mesoangioblasts. TWEAK, Fn14, and NF-κB expression was assessed by immunohistochemistry and Western blot in cell samples from both muscle biopsies and primary cultures. Mesoangioblasts isolated from samples of IBM, dermatomyositis, polymyositis, and control muscles were treated with recombinant human TWEAK, Fn14-Fc chimera, and anti-TWEAK antibody. TWEAK-RNA interference was performed in IBM and dermatomyositis mesoangioblasts. TWEAK levels in culture media were determined by enzyme-linked immunosorbent assay. In IBM muscle, we found increased TWEAK-Fn14 expression. Increased levels of TWEAK were found in differentiation medium from IBM mesoangioblasts. Moreover, TWEAK inhibited myogenic differentiation of mesoangioblasts. Consistent with this evidence, TWEAK inhibition by Fn14-Fc chimera or short interfering RNA induced myogenic differentiation of IBM mesoangioblasts. We provide evidence that TWEAK is a negative regulator of human mesoangioblast differentiation. Dysregulation of the TWEAK-Fn14 axis in IBM muscle may induce progressive muscle atrophy and reduce activation and differentiation of muscle precursor cells.