Skeletal muscle has a remarkable capacity to regenerate by virtue of its resident stem cells (satellite cells). This capacity declines with aging, although whether this is due to extrinsic changes in the environment and/or to cell-intrinsic mechanisms associated to aging has been a matter of intense debate. Furthermore, while some groups support that satellite cell aging is reversible by a youthful environment, others support cell-autonomous irreversible changes, even in the presence of youthful factors. Indeed, whereas the parabiosis paradigm has unveiled the environment as responsible for the satellite cell functional decline, satellite cell transplantation studies support cell-intrinsic deficits with aging. In this review, we try to shed light on the potential causes underlying these discrepancies. We propose that the experimental paradigm used to interrogate intrinsic and extrinsic regulation of stem cell function may be a part of the problem. The assays deployed are not equivalent and may overburden specific cellular regulatory processes and thus probe different aspects of satellite cell properties. Finally, distinct subsets of satellite cells may be under different modes of molecular control and mobilized preferentially in one paradigm than in the other. A better understanding of how satellite cells molecularly adapt during aging and their context-dependent deployment during injury and transplantation will lead to the development of efficacious compensating strategies that maintain stem cell fitness and tissue homeostasis throughout life.

BackgroundStudies of the pathogenic mechanisms underlying human myopathies and muscular dystrophies often require animal models, but models of some human diseases are not yet available. Methods to promote the engraftment and development of myogenic cells from individuals with such diseases in mice would accelerate such studies and also provide a useful tool for testing therapeutics. Here, we investigate the ability of immortalized human myogenic precursor cells (hMPCs) to form mature human myofibers following implantation into the hindlimbs of non-obese diabetic-Rag1 null IL2rγ null (NOD-Rag)-immunodeficient mice.ResultsWe report that hindlimbs of NOD-Rag mice that are X-irradiated, treated with cardiotoxin, and then injected with immortalized control hMPCs or hMPCs from an individual with facioscapulohumeral muscular dystrophy (FSHD) develop mature human myofibers. Furthermore, intermittent neuromuscular electrical stimulation (iNMES) of the peroneal nerve of the engrafted limb enhances the development of mature fibers in the grafts formed by both immortal cell lines. With control cells, iNMES increases the number and size of the human myofibers that form and promotes closer fiber-to-fiber packing. The human myofibers in the graft are innervated, fully differentiated, and minimally contaminated with murine myonuclei.ConclusionsOur results indicate that control and FSHD human myofibers can form in mice engrafted with hMPCs and that iNMES enhances engraftment and subsequent development of mature human muscle.

BackgroundSatellite cells, or muscle stem cells, have been thought to be responsible for all muscle plasticity, but recent studies using genetically modified mouse models that allow for the conditional ablation of satellite cells have challenged this dogma. Results have confirmed the absolute requirement of satellite cells for muscle regeneration but surprisingly also showed that they are not required for adult muscle growth. While the function of satellite cells in muscle growth and regeneration is becoming better defined, their role in the response to aerobic activity remains largely unexplored. The purpose of the current study was to assess the involvement of satellite cells in response to aerobic exercise by evaluating the effect of satellite cell depletion on wheel running performance.ResultsFour-month-old female Pax7/DTA mice (n = 8–12 per group) were satellite cell depleted via tamoxifen administration; at 6 months of age, mice either remained sedentary or were provided with running wheels for 8 weeks. Plantaris muscles were significantly depleted of Pax7+cells (≥90 % depleted), and 8 weeks of wheel running did not result in an increase in Pax7+ cells, or in myonuclear accretion. Interestingly, satellite cell-depleted animals ran ~27 % less distance and were 23 % slower than non-depleted animals. Wheel running was associated with elevated succinate dehydrogenase activity, muscle vascularization, lipid accumulation, and a significant shift toward more oxidative myosin heavy chain isoforms, as well as an increase in voltage dependent anion channel abundance, a marker of mitochondrial density. Importantly, these changes were independent of satellite cell content. Interestingly, depletion of Pax7+ cells from intra- as well as extrafusal muscle fibers resulted in atrophy of intrafusal fibers, thickening of muscle spindle-associated extracellular matrix, and a marked reduction of functional outcomes including grip strength, gait fluidity, and balance, which likely contributed to the impaired running performance.ConclusionsDepletion of Pax7-expressing cells in muscle resulted in reduced voluntary wheel running performance, without affecting markers of aerobic adaptation; however, their absence may perturb proprioception via disruption of muscle spindle fibers resulting in loss of gross motor coordination, indicating that satellite cells have a yet unexplored role in muscle function.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0065-3) contains supplementary material, which is available to authorized users.

BackgroundThe hormone adiponectin (ApN) is decreased in the metabolic syndrome, where it plays a key pathogenic role. ApN also exerts some anti-inflammatory effects on skeletal muscles in mice exposed to acute or chronic inflammation. Here, we investigate whether ApN could be sufficiently potent to counteract a severe degenerative muscle disease, with an inflammatory component such as Duchenne muscular dystrophy (DMD).MethodsMdx mice (a DMD model caused by dystrophin mutation) were crossed with mice overexpressing ApN in order to generate mdx-ApN mice; only littermates were used. Different markers of inflammation/oxidative stress and components of signaling pathways were studied. Global force was assessed by in vivo functional tests, and muscle injury with Evans Blue Dye (EBD). Eventually, primary cultures of human myotubes were used.ResultsCirculating ApN was markedly diminished in mdx mice. Replenishment of ApN strikingly reduced muscle inflammation, oxidative stress, and enhanced the expression of myogenic differentiation markers along with that of utrophin A (a dystrophin analog) in mdx-ApN mice. Accordingly, mdx-ApN mice exhibited higher global force and endurance as well as decreased muscle damage as quantified by curtailed extravasation of EBD in myofibers. These beneficial effects of ApN were recapitulated in human myotubes. ApN mediates its protection via the adiponectin receptor 1 (AdipoR1, the main ApN receptor in muscle) and the AMPK-SIRT1-PGC-1α signaling pathway, leading to downregulation of the nuclear factor kappa B (NF-κB) and inflammatory genes, together with upregulation of utrophin.ConclusionsAdiponectin proves to be an extremely powerful hormone capable of protecting the skeletal muscle against inflammation and injury, thereby offering novel therapeutic perspectives for dystrophinopathies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0051-9) contains supplementary material, which is available to authorized users.

BackgroundDedifferentiation, a process whereby differentiated cells lose their specialized characteristics and revert to a less differentiated state, plays a key role in the regeneration process in urodele amphibians such as the red spotted newt, Notophthalmus viridescens. Dedifferentiation of fully mature tissues is generally absent in mammalian cells. Previous studies have shown that mouse C2C12 multinucleated myotubes treated with extract derived from regenerating newt forelimbs can re-enter the cell cycle, fragment into mononucleated cells, and proliferate. However, this response has been difficult to replicate.MethodsWe isolated extract from early newt forelimb regenerates and assessed its effects on differentiation of proliferating primary and C2C12 myoblasts. We also treated fully differentiated primary and C2C12 myotube cultures with extract and assessed cell cycle re-entry and myotube fragmentation.ResultsWe have confirmed the results obtained in C2C12 cells and expanded these studies to also examine the effects of newt regeneration extracts on primary muscle cells. Newt extract can block differentiation of both C2C12 and primary myoblasts. Once differentiation is induced, treatment with newt extract causes cell cycle re-entry and fragmentation of C2C12 myotubes. Downregulation of p21 and muscle-specific markers is also induced. Primary myotubes also fragment in response to extract treatment, and the fragmented cells remain viable for long periods of time in culture. However, unlike C2C12 cells, primary muscle cells do not re-enter the cell cycle in response to treatment with newt extracts.ConclusionsDedifferentiation of fully mature muscle occurs during regeneration in the newt forelimb to contribute cells to the regeneration process. Our study shows that extracts derived from regenerating newt forelimbs can induce dedifferentiation, cell cycle re-entry, and fragmentation of mouse C2C12 cells but can only induce fragmentation in primary muscle cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0044-8) contains supplementary material, which is available to authorized users.

BackgroundThe Y-box protein MSY3/Csda represses myogenin transcription in skeletal muscle by binding a highly conserved cis-acting DNA element located just upstream of the myogenin minimal promoter (myogHCE). It is not known how this MSY3 activity is controlled in skeletal muscle. In this study, we provide multiple lines of evidence showing that the post-translational phosphorylation of MSY3 by Akt kinase modulates the MSY3 repression of myogenin.MethodsSkeletal muscle and myogenic C2C12 cells were used to study the effects of MSY3 phosphorylation in vivo and in vitro on its sub-cellular localization and activity, by blocking the IGF1/PI3K/Akt pathway, by Akt depletion and over-expression, and by mutating potential MSY3 phosphorylation sites.ResultsWe observed that, as skeletal muscle progressed from perinatal to postnatal and adult developmental stages, MSY3 protein became gradually dephosphorylated and accumulated in the nucleus. This correlated well with the reduction of phosphorylated active Akt. In C2C12 myogenic cells, blocking the IGF1/PI3K/Akt pathway using LY294002 inhibitor reduced MSY3 phosphorylation levels resulting in its accumulation in the nuclei. Knocking down Akt expression increased the amount of dephosphorylated MSY3 and reduced myogenin expression and muscle differentiation. MSY3 phosphorylation by Akt in vitro impaired its binding at the MyogHCE element, while blocking Akt increased MSY3 binding activity. While Akt over-expression rescued myogenin expression in MSY3 overexpressing myogenic cells, ablation of the Akt substrate, (Ser126 located in the MSY3 cold shock domain) promoted MSY3 accumulation in the nucleus and abolished this rescue. Furthermore, forced expression of Akt in adult skeletal muscle induced MSY3 phosphorylation and myogenin derepression.ConclusionsThese results support the hypothesis that MSY3 phosphorylation by Akt interferes with MSY3 repression of myogenin circuit activity during muscle development. This study highlights a previously undescribed Akt-mediated signaling pathway involved in the repression of myogenin expression in myogenic cells and in mature muscle. Given the significance of myogenin regulation in adult muscle, the Akt/MSY3/myogenin regulatory circuit is a potential therapeutic target to counteract muscle degenerative disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0043-9) contains supplementary material, which is available to authorized users.

BackgroundCirculating angiotensin II (AngII) is elevated in congestive heart failure (CHF), and leads to skeletal muscle wasting, which is strongly associated with poor patient outcomes. We previously found that AngII upregulates protein phosphatase 2C-alpha (PP2Cα) and dephosphorylates AMP-activated protein kinase (AMPK), a critical regulator of cellular metabolism, in skeletal muscle.MethodsTo determine the role of PP2Cα in AngII-induced wasting, gastrocnemius (Gas) muscles of FVB mice were injected with scrambled or PP2Cα siRNA and mice were infused with saline or AngII for 4 days.ResultsKnockdown of PP2Cα reduced AngII wasting, blocked AngII upregulation of PP2Cα, increased p-T172-AMPK, and inhibited AngII-mediated reductions in peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1), mitochondrial transcription factor A (TFAM), in complex IV activity, and in ATP levels. AngII impaired the rate of autophagy as determined by a 2.4-fold increase in p62/SQSTM1 (p62) accumulation. This induction was reduced by PP2Cα knockdown, which also increased beclin-1 expression and microtubule-associated protein 1 light chain 3 (LC3)-II conversion in AngII-infused Gas. AngII reduced activating S555 phosphorylation of UNC-51-like kinase 1 (ULK1), a critical regulator of autophagosome formation, and increased inhibitory S757 ULK1 phosphorylation and these effects were prevented by PP2Cα siRNA.ConclusionsAngII inhibited AMPK activity and reduced PGC-1α and TFAM expression (thereby inhibiting mitochondrial biogenesis) and impaired ULK1 activation and autophagy (thereby also inhibiting clearance of damaged mitochondria), resulting in mitochondrial dysfunction, decreased ATP, and wasting. Knockdown of PP2Cα normalized AMPK activity, PGC-1α, NRF1, and TFAM levels and blocked AngII inhibition of ULK1, leading to improved mitochondrial biogenesis/recycling/function, energy production, and inhibition of AngII-induced wasting. These results demonstrate novel effects of AngII on cellular metabolism that are likely critical in mediating the muscle wasting that is a hallmark of CHF.

BackgroundThe NFATc transcription factor family is responsible for coupling cytoplasmic calcium signals to transcription programs in a wide variety of cell types. In skeletal muscle, these transcription factors control the fiber type in response to muscle activity. This excitation-transcription (E-T) coupling permits functional adaptation of muscle according to use. The activity dependence of these transcription programs is sensitive to the firing patterns of the muscle, not merely the period of activity, enabling a nuanced adaptation to various functional tasks.MethodsIsolated skeletal muscle fibers expressing exogenous fluorescent NFATc1 were studied by confocal microscopy under stimulation both with and without pharmacological inhibitors. Western blots of whole muscle lysates were also used.ResultsThis study investigates the activity dependent response of NFATc1 skeletal muscle fibers cultured from mice, comparing fibers of respiratory origin to muscles responsible for limb locomotion. Using patterns of stimulation known to strongly activate NFATc1 in the commonly cultured flexor digitorum brevis and soleus muscles, we have observed significant deactivation of NFATc1 in cultured intercostal muscle fibers. This effect is at least partially dependent on the action of JNK and CaMKII in intercostal fibers.ConclusionsOur findings highlight the role of lineage in the NFAT pathway, showing that the respiratory intercostal muscle fibers decode similar E-T coupling signals into NFAT transcriptional programs in a different manner from the more commonly studied locomotor muscles of the limbs.

BackgroundHistological assessment of skeletal muscle tissue is commonly applied to many areas of skeletal muscle physiological research. Histological parameters including fiber distribution, fiber type, centrally nucleated fibers, and capillary density are all frequently quantified measures of skeletal muscle. These parameters reflect functional properties of muscle and undergo adaptation in many muscle diseases and injuries. While standard operating procedures have been developed to guide analysis of many of these parameters, the software to freely, efficiently, and consistently analyze them is not readily available. In order to provide this service to the muscle research community we developed an open source MATLAB script to analyze immunofluorescent muscle sections incorporating user controls for muscle histological analysis.ResultsThe software consists of multiple functions designed to provide tools for the analysis selected. Initial segmentation and fiber filter functions segment the image and remove non-fiber elements based on user-defined parameters to create a fiber mask. Establishing parameters set by the user, the software outputs data on fiber size and type, centrally nucleated fibers, and other structures. These functions were evaluated on stained soleus muscle sections from 1-year-old wild-type and mdx mice, a model of Duchenne muscular dystrophy. In accordance with previously published data, fiber size was not different between groups, but mdx muscles had much higher fiber size variability. The mdx muscle had a significantly greater proportion of type I fibers, but type I fibers did not change in size relative to type II fibers. Centrally nucleated fibers were highly prevalent in mdx muscle and were significantly larger than peripherally nucleated fibers.ConclusionsThe MATLAB code described and provided along with this manuscript is designed for image processing of skeletal muscle immunofluorescent histological sections. The program allows for semi-automated fiber detection along with user correction. The output of the code provides data in accordance with established standards of practice. The results of the program have been validated using a small set of wild-type and mdx muscle sections. This program is the first freely available and open source image processing program designed to automate analysis of skeletal muscle histological sections.