Resident Satellite Cells Research Articles

Lysine Distinctively Manipulates Myogenic Regulatory Factors and Wnt/Ca2+ Pathway in Slow and Fast Muscles, and Their Satellite Cells of Postnatal Piglets.

Muscle regeneration, representing an essential homeostatic process, relies mainly on the myogenic progress of resident satellite cells, and it is modulated by multiple physical and nutritional factors. Here, we investigated how myogenic differentiation-related factors and pathways respond to the first limiting amino acid lysine (Lys) in the fast and slow muscles, and their satellite cells (SCs), of swine. Thirty 28-day-old weaned piglets with similar body weights were subjected to three diet regimens: control group (d 0-28: 1.31% Lys, n = 12), Lys-deficient group (d 0-28: 0.83% Lys, n = 12), and Lys rescue group (d 0-14: 0.83% Lys; d 15-28: 1.31% Lys, n = 6). Pigs on d 15 and 29 were selectively slaughtered for muscular parameters evaluation. Satellite cells isolated from fast (semimembranosus) and slow (semitendinosus) muscles were also selected to investigate differentiation ability variations. We found Lys deficiency significantly hindered muscle development in both fast and slow muscles via the distinct manipulation of myogenic regulatory factors and the Wnt/Ca2+ pathway. In the SC model, Lys deficiency suppressed the Wnt/Ca2+ pathways and myosin heavy chain, myogenin, and myogenic regulatory factor 4 in slow muscle SCs but stimulated them in fast muscle SCs. When sufficient Lys was attained, the fast muscle-derived SCs Wnt/Ca2+ pathway (protein kinase C, calcineurin, calcium/calmodulin-dependent protein kinase II, and nuclear factor of activated T cells 1) was repressed, while the Wnt/Ca2+ pathway of its counterpart was stimulated to further the myogenic differentiation. Lys potentially manipulates the differentiation of porcine slow and fast muscle myofibers via the Wnt/Ca2+ pathway in opposite trends.

In Vitro Regeneration Capacity In Response To Chemical Injury Is Compromised In Aged Vs Young Muscle Cells

Healthy skeletal muscle can regenerate after ischemic, mechanical or toxin-induced injury. However, age-related changes in skeletal muscle cells and their niche may impair the regeneration potential. While strategies to regenerate muscle are being developed, in vitro models to investigate the regenerative potential after injury in aged versus young muscle cells are lacking. PURPOSE: This study aims to develop in vitro muscle models of injury and regeneration using cells from older and young donors. METHODS: Differentiated C2C12 murine and human myoblasts from old and young donors were injured after exposure to 12% barium chloride for 6 h. Regeneration was assessed by morphological analysis including myotube width, number of nuclei and fusion index. For that cells were stained with phalloidin and DAPI to label the cytoskeleton and nuclei of muscle cells, respectively. Images were acquired with the Leica fluorescence microscope and analysed using ImageJ. Timepoints were pre-injury (control), post-injury, end of proliferation and end of differentiation. RESULTS: The number of myonuclei post-injury remained unaltered compared to pre-injury among all cell lines (p > 0.05). Three days post-injury, nuclei number reached significance only in murine (p = 0.0006) and young (p = 0.03) muscle cells, whereas aged myotubes showed a significant increase in the total number of myonuclei at the end of the regeneration period (p = 0.01). Myotube diameter at the end of the regeneration recovered to pre-injury state in C2C12 and young but was significantly smaller in aged cells (p = 0.04). CONCLUSION: Delayed peak in proliferation and significantly smaller myotubes in aged compared to young and murine muscle cells at the end of the experiment could be attributed to declined proliferation rate of resident satellite cells that causes suspended myogenesis and regenerative response in older muscle cells. Barium chloride injury model is an easily reproducible in vitro approach that could be applied to study age and/or disease-related regenerative changes in different cell lines. KEYWORDS: regeneration, injury, skeletal muscle, ageing

In Vitro Model of Human Skeletal Muscle Tissue for the Study of Resident Macrophages and Stem Cells.

Simple SummaryThe skeletal muscle of younger adults has a remarkable regenerative capacity, which substantially declines with age. Despite many interspecies differences, animals have been used to study new treatments to promote muscle regeneration in humans. This study reports a novel human experimental model using human skeletal muscle tissue of older adults that was extracted during surgical procedures. We describe an optimal procedure for maintaining human skeletal muscle tissue under experimental conditions for 11 days. This experimental model allows the investigation of resident macrophages and stem cells, which mediate muscle regeneration.Findings from studies of muscle regeneration can significantly contribute to the treatment of age-related loss of skeletal muscle mass, which may predispose older adults to severe morbidities. We established a human experimental model using excised skeletal muscle tissues from reconstructive surgeries in eight older adults. Muscle samples from each participant were preserved immediately or maintained in agarose medium for the following 5, 9, or 11 days. Immunofluorescence analyses of the structural proteins, actin and desmin, confirmed the integrity of muscle fibers over 11 days of maintenance. Similarly, the numbers of CD80-positive M1 and CD163-positive M2 macrophages were stable over 11 days in vitro. However, the numbers of PAX7-positive satellite cells and MYOD-positive myoblasts changed in opposite ways, suggesting that satellite cells partially differentiated in vitro. Further experiments revealed that stimulation with unsaturated fatty acid C18[2]c (linoleic acid) increased resident M1 macrophages and satellite cells specifically. Thus, the use of human skeletal muscle tissue in vitro provides a direct experimental approach to study the regulation of muscle tissue regeneration by macrophages and stem cells and their responses to therapeutic compounds.

Human muscle stem cells are refractory to aging.

Age‐related loss of muscle mass and strength is widely attributed to limitation in the capacity of muscle resident satellite cells to perform their myogenic function. This idea contains two notions that have not been comprehensively evaluated by experiment. First, it entails the idea that we damage and lose substantial amounts of muscle in the course of our normal daily activities. Second, it suggests that mechanisms of muscle repair are in some way exhausted, thus limiting muscle regeneration. A third potential option is that the aged environment becomes inimical to the conduct of muscle regeneration. In the present study, we used our established model of human muscle xenografting to test whether muscle samples taken from cadavers, of a range of ages, maintained their myogenic potential after being transplanted into immunodeficient mice. We find no measurable difference in regeneration across the range of ages investigated up to 78 years of age. Moreover, we report that satellite cells maintained their myogenic capacity even when muscles were grafted 11 days postmortem in our model. We conclude that the loss of muscle mass with increasing age is not attributable to any intrinsic loss of myogenicity and is most likely a reflection of progressive and detrimental changes in the muscle microenvironment such as to disfavor the myogenic function of these cells.

Skeletal muscle regeneration via the chemical induction and expansion of myogenic stem cells in situ or in vitro.

Muscle loss and impairment resulting from traumatic injury can be alleviated by therapies using muscle stem cells. However, harvesting sufficient numbers of autologous myogenic stem cells and expanding them efficiently has been challenging. Here, we show that myogenic stem cells (predominantly Pax7+ cells) selectively expanded from readily obtainable dermal fibroblasts or skeletal muscle stem cells via a specific cocktail of small molecules and transplanted into muscle injuries in adult, aged or dystrophic mice led to functional muscle regeneration in the three animal models, and that sustained release of the small-molecule cocktail in situ via polymer nanoparticles also led to muscle repair by inducing the robust activation and expansion of resident satellite cells. Chemically induced stem cell expansion in vitro and in situ may prove advantageous for stem cell therapies aiming to regenerate skeletal muscle and other tissues.

Lgr5 Marks Adult Progenitor Cells Contributing to Skeletal Muscle Regeneration and Sarcoma Formation

Regeneration of adult skeletal muscle is driven largely by resident satellite cells, a stem cell population increasingly considered to display a high degree of molecular heterogeneity. In this study, we find that Lgr5, a receptor for Rspo and a potent mediator of Wnt/β-catenin signaling, marks a subset of activated satellite cells that contribute to muscle regeneration. Lgr5 is found to be rapidly upregulated in purified myogenic progenitors following acute cardiotoxin-induced injury. Invivo lineage tracing using our Lgr5-2ACreERT2R26tdTomatoLSL reporter mouse model shows that Lgr5+ cells can reconstitute damaged muscle fibers following muscle injury, as well as replenish the quiescent satellite cell pool. Moreover, conditional mutation in Lgr52ACreERT2;KrasG12D;Trp53flox/flox mice drives undifferentiated pleomorphic sarcoma formation in adult mice, thereby substantiating Lgr5+ cells as a cell of origin of sarcomas. Our findings provide the groundwork for developing Rspo/Wnt-signaling-based therapeutics to potentially enhance regenerative outcomes of skeletal muscles in degenerative muscle diseases.

Skeletal Muscle Tissue Engineering: Biomaterials-Based Strategies for the Treatment of Volumetric Muscle Loss.

Millions of Americans suffer from skeletal muscle injuries annually that can result in volumetric muscle loss (VML), where extensive musculoskeletal damage and tissue loss result in permanent functional deficits. In the case of small-scale injury skeletal muscle is capable of endogenous regeneration through activation of resident satellite cells (SCs). However, this is greatly reduced in VML injuries, which remove native biophysical and biochemical signaling cues and hinder the damaged tissue’s ability to direct regeneration. The current clinical treatment for VML is autologous tissue transfer, but graft failure and scar tissue formation leave patients with limited functional recovery. Tissue engineering of instructive biomaterial scaffolds offers a promising approach for treating VML injuries. Herein, we review the strategic engineering of biophysical and biochemical cues in current scaffold designs that aid in restoring function to these preclinical VML injuries. We also discuss the successes and limitations of the three main biomaterial-based strategies to treat VML injuries: acellular scaffolds, cell-delivery scaffolds, and in vitro tissue engineered constructs. Finally, we examine several innovative approaches to enhancing the design of the next generation of engineered scaffolds to improve the functional regeneration of skeletal muscle following VML injuries.

Tumor Necrosis Factor Alpha Regulates Skeletal Myogenesis by Inhibiting SP1 Interaction with cis-Acting Regulatory Elements within the Fbxl2 Gene Promoter.

FBXL2 is an important ubiquitin E3 ligase component that modulates inflammatory signaling and cell cycle progression, but its molecular regulation is largely unknown. Here, we show that tumor necrosis factor alpha (TNF-α), a critical cytokine linked to the inflammatory response during skeletal muscle regeneration, suppressed Fbxl2 mRNA expression in C2C12 myoblasts and triggered significant alterations in cell cycle, metabolic, and protein translation processes. Gene silencing of Fbxl2 in skeletal myoblasts resulted in increased proliferative responses characterized by activation of mitogen-activated protein (MAP) kinases and nuclear factor kappa B and decreased myogenic differentiation, as reflected by reduced expression of myogenin and impaired myotube formation. TNF-α did not destabilize the Fbxl2 transcript (half-life [t1/2], ∼10 h) but inhibited SP1 transactivation of its core promoter, localized to bp -160 to +42 within the proximal 5' flanking region of the Fbxl2 gene. Chromatin immunoprecipitation and gel shift studies indicated that SP1 interacted with the Fbxl2 promoter during cellular differentiation, an effect that was less pronounced during proliferation or after TNF-α exposure. TNF-α, via activation of JNK, mediated phosphorylation of SP1 that impaired its binding to the Fbxl2 promoter, resulting in reduced transcriptional activity. The results suggest that SP1 transcriptional activation of Fbxl2 is required for skeletal muscle differentiation, a process that is interrupted by a key proinflammatory myopathic cytokine.IMPORTANCE Skeletal muscle regeneration and repair involve the recruitment and proliferation of resident satellite cells that exit the cell cycle during the process of myogenic differentiation to form myofibers. We demonstrate that the ubiquitin E3 ligase subunit FBXL2 is essential for skeletal myogenesis through its important effects on cell cycle progression and cell proliferative signaling. Further, we characterize a new mechanism whereby sustained stimulation by a major proinflammatory cytokine, TNF-α, regulates skeletal myogenesis by inhibiting the interaction of SP1 with the Fbxl2 core promoter in proliferating myoblasts. Our findings contribute to the understanding of skeletal muscle regeneration through the identification of Fbxl2 as both a critical regulator of myogenic proliferative processes and a susceptible gene target during inflammatory stimulation by TNF-α in skeletal muscle. Modulation of Fbxl2 activity may have relevance to disorders of muscle wasting associated with sustained proinflammatory signaling.

Barium chloride injures myofibers through calcium-induced proteolysis with fragmentation of motor nerves and microvessels

BackgroundLocal injection of BaCl2 is an established model of acute injury to study the regeneration of skeletal muscle. However, the mechanism by which BaCl2 causes muscle injury is unresolved. Because Ba2+ inhibits K+ channels, we hypothesized that BaCl2 induces myofiber depolarization leading to Ca2+ overload, proteolysis, and membrane disruption. While BaCl2 spares resident satellite cells, its effect on other tissue components integral to contractile function has not been defined. We therefore asked whether motor nerves and microvessels, which control and supply myofibers, are injured by BaCl2 treatment.MethodsThe intact extensor digitorum longus (EDL) muscle was isolated from male mice (aged 3–4 months) and irrigated with physiological salt solution (PSS) at 37 °C. Myofiber membrane potential (Vm) was recorded using sharp microelectrodes while intracellular calcium concentration ([Ca2+]i) was evaluated with Fura 2 dye. Isometric force production of EDL was measured in situ, proteolytic activity was quantified by calpain degradation of αII-spectrin, and membrane disruption was marked by nuclear staining with propidium iodide (PI). To test for effects on motor nerves and microvessels, tibialis anterior or gluteus maximus muscles were injected with 1.2% BaCl2 (50–75 μL) in vivo followed by immunostaining to evaluate the integrity of respective tissue elements post injury. Data were analyzed using Students t test and analysis of variance with P ≤ 0.05 considered statistically significant.ResultsAddition of 1.2% BaCl2 to PSS depolarized myofibers from − 79 ± 3 mV to − 17 ± 7 mV with a corresponding rise in [Ca2+]i; isometric force transiently increased from 7.4 ± 0.1 g to 11.1 ± 0.4 g. Following 1 h of BaCl2 exposure, 92 ± 3% of myonuclei stained with PI (vs. 8 ± 3% in controls) with enhanced cleavage of αII-spectrin. Eliminating Ca2+ from PSS prevented the rise in [Ca2+]i and ameliorated myonuclear staining with PI during BaCl2 exposure. Motor axons and capillary networks appeared fragmented within 24 h following injection of 1.2% BaCl2 and morphological integrity deteriorated through 72 h.ConclusionsBaCl2 injures myofibers through depolarization of the sarcolemma, causing Ca2+ overload with transient contraction, leading to proteolysis and membrane rupture. Motor innervation and capillarity appear disrupted concomitant with myofiber damage, further compromising muscle integrity.

Biomimetic sponges for regeneration of skeletal muscle following trauma.

Skeletal muscle is inept in regenerating after traumatic injuries due to significant loss of basal lamina and the resident satellite cells. To improve regeneration of skeletal muscle, we have developed biomimetic sponges composed of collagen, gelatin, and laminin (LM)-111 that were crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). Collagen and LM-111 are crucial components of the muscle extracellular matrix and were chosen to impart bioactivity whereas gelatin and EDC were used to provide mechanical strength to the scaffold. Morphological and mechanical evaluation of the sponges showed porous structure, water-retention capacity and a compressive modulus of 590-808 kPa. The biomimetic sponges supported the infiltration and viability of C2 C12 myoblasts over 5 days of culture. The myoblasts produced higher levels of myokines such as VEGF, IL-6, and IGF-1 and showed higher expression of myogenic markers such as MyoD and myogenin on the biomimetic sponges. Biomimetic sponges implanted in a mouse model of volumetric muscle loss (VML) supported satellite, endothelial, and inflammatory cell infiltration but resulted in limited myofiber regeneration at 2 weeks post-injury. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 92-103, 2019.

Senescence in Myoblasts; Role in Age‐Related Muscle Dysfunction

Loss of skeletal muscle mass and function are critical factors in the development of frailty although the mechanisms by which this loss occurs are poorly understood. The number of senescent cells increases with age in rodents (Krishnamurthy et al., 2004) and primates (Kreiling et al., 2011) and it is proposed that the presence of these cells plays a role in age‐related muscle dysfunction. We hypothesise that resident satellite cells become senescent and exert detrimental effects on mature muscle fibres via the senescence associated secretory phenotype molecules (SASP).Initial studies have investigated whether a C2C12 mouse muscle myoblast cell line could be induced into senescence, whether key senescence associated molecules (such as p16, p21, and p53) are upregulated and determined the nature of the SASP.C2C12 myoblasts were treated for 24 hours with the topoisomerase II inhibitor, Etoposide, to cause DNA damage. Myoblasts were examined for up to 5 days following treatment for cell viability, Senescence Associated β‐Galactosidase (SAβ‐Gal) activity, immunocytochemistry and western blot analysis for p16, p21, and p53.Treated myoblasts exhibited a significantly altered morphology consistent with a senescence phenotype, increased SAβ‐Gal activity, and cyclin‐dependent kinase inhibitor levels compared with untreated cells.These data suggest that myoblasts can be induced into a senescent phenotype. Further work will characterise the SASP of the cells and determine the effect on mature myotubes co‐cultured with senescent myoblasts.Support or Funding InformationWork generously funded by the British Society for Research into Ageing and the Rosetrees Trust.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Skeletal muscle regeneration involves macrophage-myoblast bonding

ABSTRACTRegeneration in adult skeletal muscle relies on the activation, proliferation, and fusion of myogenic precursor cells (MPC), mostly resident satellite cells (SC). However, the regulatory mechanism during this process is still under evaluation, with the final aim to manipulate regeneration when the intrinsic mechanism is corrupted. Furthermore, intercellular connections during skeletal muscle regeneration have not been previously thoroughly documented. Our hypothesis was that a direct and close cellular interaction between SC/MPC and invading myeloid cells is a key step to control regeneration. We tested this hypothesis during different steps of skeletal muscle regeneration: (a) the recruitment of activated SC; (b) the differentiation of MPC; (c) myotubes growth, in a mouse model of crush injury. Samples harvested (3 and 5 days) post-injury were screened by light and confocal microscopy. Ultrastructural analysis was performed by conventional transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) followed by 3D modeling of electron tomography (ET) data. This revealed a new type of interaction between macrophages and myogenic cells by direct heterocellular surface apposition over large areas and long linear distances. In the analyzed volume, regions spaced below 20 nm, within molecular range, represented 31% of the macrophage membrane surface and more than 27% of the myotube membrane. The constant interaction throughout all stages of myogenesis suggests a potential new type of regulatory mechanism for the myogenic process. Thus, deciphering structural and molecular mechanisms of SC-macrophage interaction following injury might open promising perspectives for improving muscle healing.

Identification and Characterization of the Dermal Panniculus Carnosus Muscle Stem Cells.

SummaryThe dermal Panniculus carnosus (PC) muscle is important for wound contraction in lower mammals and represents an interesting model of muscle regeneration due to its high cell turnover. The resident satellite cells (the bona fide muscle stem cells) remain poorly characterized. Here we analyzed PC satellite cells with regard to developmental origin and purported function. Lineage tracing shows that they originate in Myf5+, Pax3/Pax7+ cell populations. Skin and muscle wounding increased PC myofiber turnover, with the satellite cell progeny being involved in muscle regeneration but with no detectable contribution to the wound-bed myofibroblasts. Since hematopoietic stem cells fuse to PC myofibers in the absence of injury, we also studied the contribution of bone marrow-derived cells to the PC satellite cell compartment, demonstrating that cells of donor origin are capable of repopulating the PC muscle stem cell niche after irradiation and bone marrow transplantation but may not fully acquire the relevant myogenic commitment.

Influence of exercise and aging on extracellular matrix composition in the skeletal muscle stem cell niche.

Skeletal muscle is endowed with a remarkable capacity for regeneration, primarily due to the reserve pool of muscle resident satellite cells. The satellite cell is the physiologically quiescent muscle stem cell that resides beneath the basal lamina and adjacent to the sarcolemma. The anatomic location of satellite cells is in close proximity to vasculature where they interact with other muscle resident stem/stromal cells (e.g., mesenchymal stem cells and pericytes) through paracrine mechanisms. This mini-review describes the components of the muscle stem cell niche, as well as the influence of exercise and aging on the muscle stem cell niche. Although exercise promotes ECM reorganization and stem cell accumulation, aging is associated with dense ECM deposition and loss of stem cell function resulting in reduced regenerative capacity and strength. An improved understanding of the niche elements will be valuable to inform the development of therapeutic interventions aimed at improving skeletal muscle regeneration and adaptation over the life span.

Flow Cytometry and Transplantation-Based Quantitative Assays for Satellite Cell Self-Renewal and Differentiation.

In response to muscle damage, satellite cells proliferate and undertake both differentiation and self-renewal, generating new functional muscle tissue and repopulating this new muscle with stem cells for future injury responses. For many questions relating to the physiological regulation of satellite cells, quantitative readouts of self-renewal and differentiation can be very useful. There is a particular need for a quantitative assay for satellite cell self-renewal that does not rely solely upon sectioning, staining and counting cells in sections. In this chapter, we provide detailed methods for quantifying the self-renewal and differentiation potential of a given population of satellite cells using an assay involving transplantation into injured, regenerating muscle together with specific markers for donor cell identity and state of differentiation. In particular, using the Pax7-ZsGreen transgene as a marker of satellite cell state, self-renewal can be quantified by FACS on transplanted muscle to actually count the total number of resident satellite cells at time points following transplantation.

Cellular players in skeletal muscle regeneration.

Skeletal muscle, a tissue endowed with remarkable endogenous regeneration potential, is still under focused experimental investigation mainly due to treatment potential for muscle trauma and muscular dystrophies. Resident satellite cells with stem cell features were enthusiastically described quite a long time ago, but activation of these cells is not yet controlled by any medical interventions. However, after thorough reports of their existence, survival, activation, and differentiation there are still many questions to be answered regarding the intimate mechanism of tissue regeneration. This review delivers an up-to-date inventory of the main known key players in skeletal muscle repair, revealed by various models of tissue injuries in mechanical trauma, toxic lesions, and muscular dystrophy. A better understanding of the spatial and temporal relationships between various cell populations, with different physical or paracrine interactions and phenotype changes induced by local or systemic signalling, might lead to a more efficient approach for future therapies.

Enhanced Satellite Cell Activity in Aging Skeletal Muscle after Manual Acupuncture-Induced Injury

Skeletal muscle injury stimulates normally quiescent resident satellite (stem) cells to re-enter the cell cycle and execute the myogenic program to restore muscle structure and function. Previously, we reported that manual acupuncture needling of the tibialis anterior (TA) (ST36 = Zusanli) muscle of young male rats produced focal injury and morphological changes that accompanied the presence of activated satellite cells (SC) 72 hours post-needling. To investigate whether aging TA muscle responds in a similar fashion to acupuncture needling, 17-month-old female rats were subjected to a single insertion and manual manipulation of an acupuncture needle. At 72 hours’ post-needling, hematoxylin staining of the TA revealed increased mononuclear cell infiltration that was indicative of localized injury. Moreover, this was accompanied by a four-fold increase in the expression of proliferating cell nuclear antigen within cells of needled tissues. Heightened immunofluorescence for MyoD was found within SC in the needled muscle, which correlated with a 6- and 10-fold increase in two MyoD isoforms (~38 and 42 kDa, respectively), when analyzed by Western blotting. An additional 56 kDa MyoD immunoreactive species was observed in both needled and control muscle of the aging rats. The present study in pre-senile female rats, in conjunction with our previous study in young male rats, suggests that muscle remodeling and restructuring after injury may constitute the initial cellular and molecular mechanisms that underlie the benefits associated with acupuncture throughout the life-span.

A novel in vitro model for studying quiescence and activation of primary isolated human myoblasts.

Skeletal muscle stem cells, satellite cells, are normally quiescent but become activated upon muscle injury. Recruitment of resident satellite cells may be a useful strategy for treatment of muscle disorders, but little is known about gene expression in quiescent human satellite cells or the mechanisms involved in their early activation. We have developed a method to induce quiescence in purified primary human myoblasts isolated from healthy individuals. Analysis of the resting state showed absence of BrdU incorporation and lack of KI67 expression, as well as the extended kinetics during synchronous reactivation into the cell cycle, confirming arrest in the G0 phase. Reactivation studies showed that the majority (>95%) of the G0 arrested cells were able to re-enter the cell cycle, confirming reversibility of arrest. Furthermore, a panel of important myogenic factors showed expression patterns similar to those reported for mouse satellite cells in G0, reactivated and differentiated cultures, supporting the applicability of the human model. In addition, gene expression profiling showed that a large number of genes (4598) were differentially expressed in cells activated from G0 compared to long term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis of quiescence and reactivation of human myoblasts.

Activation and nuclear translocation of PKCε promotes skeletal muscle cell differentiation via HMGA1 downregulation

The role of novel PKCs in skeletal muscle differentiation has recently emerged. PKCθ is the most expressed isoform of PKCs in muscle and it promotes the fusion of myoblasts [1]. Recently, we have demonstrated that PKCe is implicated in myocardiocyte differentiation of bone marrow mesenchymal stem cells [2] but the role of PKCe in skeletal muscle cell regeneration has only recently emerged [3]. We here demonstrate that both nuclear and cytoplasmic fractions of PKCe are up-regulated during in vitro C2C12 cell line and satellite cell differentiation. We also show that PKCe is able to modulate myogenic differentiation genes via a downmodulation of HMGA1 proteins that promotes myogenin accumulation and mature myoblast formation. To study the effects of PKCe on muscle regeneration, we have used the in vivo model of CTX-induced skeletal muscle injury. We show that the upregulation of PKCe also occurs in vivo, particularly in the centro-nucleated regenerating fibers that are derived from the fusion process of the resident satellite cells, suggesting a role for PKCe in human satellite cell-driven muscle repair and substitution, with clinically relevant implications in human muscle pathology.

Compromised genomic integrity impedes muscle growth after Atrx inactivation

ATR-X syndrome is a severe intellectual disability disorder caused by mutations in the ATRX gene. Many ancillary clinical features are attributed to CNS deficiencies, yet most patients have muscle hypotonia, delayed ambulation, or kyphosis, pointing to an underlying skeletal muscle defect. Here, we identified a cell-intrinsic requirement for Atrx in postnatal muscle growth and regeneration in mice. Mice with skeletal muscle-specific Atrx conditional knockout (Atrx cKO mice) were viable, but by 3 weeks of age presented hallmarks of underdeveloped musculature, including kyphosis, 20% reduction in body mass, and 34% reduction in muscle fiber caliber. Atrx cKO mice also demonstrated a marked regeneration deficit that was not due to fewer resident satellite cells or their inability to terminally differentiate. However, activation of Atrx-null satellite cells from isolated muscle fibers resulted in a 9-fold reduction in myoblast expansion, caused by delayed progression through mid to late S phase. While in S phase, Atrx colocalized specifically to late-replicating chromatin, and its loss resulted in rampant signs of genomic instability. These observations support a model in which Atrx maintains chromatin integrity during the rapid developmental growth of a tissue.