The role of Pitx2 and Pitx3 in muscle stem cells gives new insights into P38α MAP kinase and redox regulation of muscle regeneration.

Skeletal muscle regeneration depends on satellite cells. After injury these muscle stem cells exit quiescence, proliferate and differentiate to regenerate damaged fibres. We show that this progression is accompanied by metabolic changes leading to increased production of reactive oxygen species (ROS). Using Pitx2/3 single and double mutant mice that provide genetic models of deregulated redox states, we demonstrate that moderate overproduction of ROS results in premature differentiation of satellite cells while high levels lead to their senescence and regenerative failure. Using the ROS scavenger, N-Acetyl-Cysteine (NAC), in primary cultures we show that a physiological increase in ROS is required for satellite cells to exit the cell cycle and initiate differentiation through the redox activation of p38α MAP kinase. Subjecting cultured satellite cells to transient inhibition of P38α MAP kinase in conjunction with NAC treatment leads to their rapid expansion, with striking improvement of their regenerative potential in grafting experiments.

Dynamic Phosphorylation of the Myocyte Enhancer Factor 2Cα1 Splice Variant Promotes Skeletal Muscle Regeneration and Hypertrophy.

The transcription factor MEF2C (Myocyte Enhancer Factor 2C) plays an established role in the early steps of myogenic differentiation. However, the involvement of MEF2C in adult myogenesis and in muscle regeneration has not yet been systematically investigated. Alternative splicing of mammalian MEF2C transcripts gives rise to two mutually exclusive protein variants: MEF2Cα2 which exerts a positive control of myogenic differentiation, and MEF2Cα1, in which the α1 domain acts as trans-repressor of the MEF2C pro-differentiation activity itself. However, MEF2Cα1 variants are persistently expressed in differentiating cultured myocytes, suggesting a role in adult myogenesis. We found that overexpression of both MEF2Cα1/α2 proteins in a mouse model of muscle injury promotes muscle regeneration and hypertrophy, with each isoform promoting different stages of myogenesis. Besides the ability of MEF2Cα2 to increase differentiation, we found that overexpressed MEF2Cα1 enhances both proliferation and differentiation of primary myoblasts, and activates the AKT/mTOR/S6K anabolic signaling pathway in newly formed myofibers. The multiple activities of MEF2Cα1 are modulated by phosphorylation of Ser98 and Ser110, two amino acid residues located in the α1 domain of MEF2Cα1. These specific phosphorylations allow the interaction of MEF2Cα1 with the peptidyl-prolyl isomerase PIN1, a regulator of MEF2C functions. Overall, in this study we established a novel regulatory mechanism in which the expression and the phosphorylation of MEF2Cα1 are critically required to sustain the adult myogenesis. The described molecular mechanism will represent a new potential target for the development of therapeutical strategies to treat muscle-wasting diseases. Stem Cells 2017;35:725-738.

Endothelial cell specification in the somite is compromised in Pax3-positive progenitors of Foxc1/2 conditional mutants, with loss of forelimb myogenesis.

Pax3 and Foxc2 have been shown genetically to mutually repress each other in the mouse somite. Perturbation of this balance in multipotent cells of the dermomyotome influences cell fate; upregulation of Foxc2 favours a vascular fate, whereas higher levels of Pax3 lead to myogenesis. Foxc1 has overlapping functions with Foxc2. In Foxc1/2 double-mutant embryos, somitogenesis is severely affected, precluding analysis of somite derivatives. We have adopted a conditional approach whereby mutations in Foxc1 and Foxc2 genes were targeted to Pax3-expressing cells. Inclusion of a conditional reporter allele in the crosses made it possible to follow cells that had expressed Pax3. At the forelimb level, endothelial and myogenic cells migrate from adjacent somites into the limb bud. This population of endothelial cells is compromised in the double mutant, whereas excessive production of myogenic cells is observed in the trunk. However, strikingly, myogenic progenitors fail to enter the limbs, leading to the absence of skeletal muscle. Pax3-positive migratory myogenic progenitors, marked by expression of Lbx1, are specified in the somite at forelimb level, but endothelial progenitors are absent. The myogenic progenitors do not die, but differentiate prematurely adjacent to the somite. We conclude that the small proportion of somite-derived endothelial cells in the limb is required for the migration of myogenic limb progenitors.

Redox regulation by Pitx2 and Pitx3 is critical for fetal myogenesis.

During development, major metabolic changes occur as cells become more specialized within a lineage. In the case of skeletal muscle, differentiation is accompanied by a switch from a glycolytic proliferative progenitor state to an oxidative postmitotic differentiated state. Such changes require extensive mitochondrial biogenesis leading to increased reactive oxygen species (ROS) production that needs to be balanced by an antioxidant system. Our analysis of double conditional Pitx2/3 mouse mutants, both in vivo during fetal myogenesis and ex vivo in primary muscle cell cultures, reveals excessive upregulation of ROS levels leading to DNA damage and apoptosis of differentiating cells. This is a consequence of downregulation of Nrf1 and genes for antioxidant enzymes, direct targets of Pitx2/3, leading to decreased expression of antioxidant enzymes, as well as impairment of mitochondrial function. Our analysis identifies Pitx2 and Pitx3 as key regulators of the intracellular redox state preventing DNA damage as cells undergo differentiation.

Pitx2 and Pitx3 transcription factors: two key regulators of the redox state in adult skeletal muscle stem cells and muscle regeneration.

Adult tissue homeostasis and regeneration rely on tissue stem cell populations that generate committed precursors and differentiated cells while maintaining a pool of stem cells. In adult skeletal muscle, such cells, called satellite cells, remain quiescent at the periphery of muscle fibers. Upon injury they undergo activation, proliferation and differentiation to replace damaged fibers and also self-renew to reconstitute the muscle stem cell pool. During regeneration, the transition from a quiescent muscle stem cell to a differentiated fiber is accompanied by major metabolic changes. Such changes, and notably the switch from a glycolytic proliferative progenitor state to an oxidative post-mitotic differentiated state, require extensive mitochondrial biogenesis that takes place at the onset of differentiation and leads to increased ROS production. However, it is unclear whether this enhanced ROS production/mitochondrial content reflects an adaptation to the rising energy demand or whether it constitutes an essential regulation element of the differentiation program.To investigate the potential role of this metabolic switch and more specifically of reactive oxygen species during muscle regeneration, we took advantage of mouse mutants for Pitx2 and Pitx3 genes. Both genes are involved in foetal myogenesis where they have been identified as key regulators of the redox state preventing excessive ROS levels and DNA damage as cells undergo differentiation. We have now analyzed adult single and double Pitx2:Pitx3 conditional mutant mouse lines targeted to the muscle stem cell compartment. Double mutant satellite cells undergo senescence with impaired regeneration after injury, whereas in single Pitx3 mutants, premature differentiation occurs. We show that these effects are directly linked to dose-dependent changes in ROS levels and can be reversed by lowering ROS with the N-acetylcystein, supporting the notion that a controlled increase in ROS is required for differentiation of muscle stem cells.

Lying low but ready for action: the quiescent muscle satellite cell.

The muscle satellite cell is essential for skeletal muscle regeneration. It is located on the muscle fibre, under the basal lamina as a quiescent cell, which becomes activated after injury, when it leaves the fibre, proliferates, and either undergoes myogenesis to form new fibres or reconstitutes the satellite cell pool. In this review, we discuss the cellular environment of the quiescent cell, including the extracellular matrix, which constitutes its niche. Cell adhesion molecules and some signalling pathways reinforce its quiescent state, whereas other signals lead to activation. We discuss how the satellite cell is ready to respond with the appropriate receptors, but protects its quiescence by mechanisms that include immobilization of ligands by extracellular matrix components and synthesis of inhibitors for intracellular signalling pathways and for metalloproteinases that break down the matrix and promote ligand processing and receptor activation. The quiescent satellite cell is also well protected against toxins and oxidative stress. It has a low metabolic rate, as shown by few active mitochondria and anaerobic glycolysis. Different subpopulations of quiescent satellite cells can be distinguished on the basis of cell surface markers and stem cell-like properties. We discuss the latter in the context of the small proportion of satellite cells that express high levels of Pax7, or that are derived from cells that have never activated the Myf5 myogenic determination gene. However, many quiescent satellite cells transcribe Myf5, but do not enter myogenesis because of post-transcriptional regulation, which prevents Myf5 protein accumulation. Post-transcriptional regulation, through microRNA repression of a potential cell cycle activator, further illustrates how these cells are ready for action.

Itm2a is a Pax3 target gene, expressed at sites of skeletal muscle formation in vivo.

The paired-box homeodomain transcription factor Pax3 is a key regulator of the nervous system, neural crest and skeletal muscle development. Despite the important role of this transcription factor, very few direct target genes have been characterized. We show that Itm2a, which encodes a type 2 transmembrane protein, is a direct Pax3 target in vivo, by combining genetic approaches and in vivo chromatin immunoprecipitation assays. We have generated a conditional mutant allele for Itm2a, which is an imprinted gene, by flanking exons 2-4 with loxP sites and inserting an IRESnLacZ reporter in the 3' UTR of the gene. The LacZ reporter reproduces the expression profile of Itm2a, and allowed us to further characterize its expression at sites of myogenesis, in the dermomyotome and myotome of somites, and in limb buds, in the mouse embryo. We further show that Itm2a is not only expressed in adult muscle fibres but also in the satellite cells responsible for regeneration. Itm2a mutant mice are viable and fertile with no overt phenotype during skeletal muscle formation or regeneration. Potential compensatory mechanisms are discussed.

Fetal skeletal muscle progenitors have regenerative capacity after intramuscular engraftment in dystrophin deficient mice.

Muscle satellite cells (SCs) are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs) isolated from Pax3(GFP/+) embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3(GFP/+) mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease.

Myod and H19-Igf2 locus interactions are required for diaphragm formation in the mouse.

The myogenic regulatory factor Myod and insulin-like growth factor 2 (Igf2) have been shown to interact in vitro during myogenic differentiation. In order to understand how they interact in vivo, we produced double-mutant mice lacking both the Myod and Igf2 genes. Surprisingly, these mice display neonatal lethality due to severe diaphragm atrophy. Alteration of diaphragm muscle development occurs as early as 15.5 days post-coitum in the double-mutant embryos and leads to a defect in the terminal differentiation of muscle progenitor cells. A negative-feedback loop was detected between Myod and Igf2 in embryonic muscles. Igf2 belongs to the imprinted H19-Igf2 locus. Molecular analyses show binding of Myod on a mesodermal enhancer (CS9) of the H19 gene. Chromatin conformation capture experiments reveal direct interaction of CS9 with the H19 promoter, leading to increased H19 expression in the presence of Myod. In turn, the non-coding H19 RNA represses Igf2 expression in trans. In addition, Igf2 also negatively regulates Myod expression, possibly by reducing the expression of the Srf transcription factor, a known Myod activator. In conclusion, Igf2 and Myod are tightly co-regulated in skeletal muscles and act in parallel pathways in the diaphragm, where they affect the progression of myogenic differentiation. Igf2 is therefore an essential player in the formation of a functional diaphragm in the absence of Myod.

Altered in vitro proliferation of mouse SOD1-G93A skeletal muscle satellite cells.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is the most common adult-onset neurodegenerative disease characterized by ascending muscle weakness, atrophy and paralysis. Early muscle abnormalities that precede motor neuron loss in ALS may destabilize neuromuscular junctions, and we have previously demonstrated alterations in myogenic regulatory factor (MRF) expression in vivo and in the activation of myofiber-associated skeletal muscle satellite cells (SMSCs) in the mouse model of ALS (SOD1-G93A). METHODS: To elucidate niche dependence versus cell-autonomous mutant SOD1 (mSOD1) toxicity in this model, we measured in vitro proliferation potential and MRF and cyclin gene expression in SMSC cultures derived from fast-twitch extensor digitorum longus and slow-twitch soleus muscles of SOD1-G93A mice. RESULTS: SMSCs from early presymptomatic (p40) to terminal, semi-paralytic (p120) SOD1-G93A mice demonstrated generally lower proliferation potential compared with age-matched controls. However, induced proliferation was observed in surgically denervated wild-type animals and SOD1-G93A animals at p90, when critical denervation arises. SMSCs from fast and slow muscles were similarly affected by mSOD1 expression. Lowered proliferation rate was generally corroborated with decreased relative MRF expression levels, although this was most prominent in early age and was modulated by muscle type origin. Cyclins controlling cell proliferation did not show modifications in their mRNA levels; however, the expression of cyclin-dependent kinase inhibitor 1A (Cdkn1a), which is known to promote myoblast differentiation, was decreased in SOD1-G93A cultures. CONCLUSIONS: Our data suggest that the function of SMSCs is impaired in SOD1-G93A satellite cells from the earliest stages of the disease when no critical motor neuron loss has been described.

Double Myod and Igf2 inactivation promotes brown adipose tissue development by increasing Prdm16 expression.

Brown fat or brown adipose tissue (BAT), found in newborn mammals as small depots localized in the interscapular region, plays a prominent role in regulating thermogenesis perinatally. The physiological importance of functional BAT has been recently reasserted in human adults. Because myoblasts and adipoblasts emerge from a common mesodermal precursor, we investigated developmental determination and the reciprocal relationship between muscle and adipocyte commitment. Here we show that a mutant mouse defective for both Igf2 and Myod genes exhibits massive BAT hypertrophy compared with wild-type and single-mutant newborns. The increased adipocyte proliferation in BAT of double-mutant newborns was associated with overexpression of the brown fat-specific marker Ucp1. More strikingly, expression of the master key gene Prdm16 involved in the switch between myogenic and brown adipogenic lineages was drastically enhanced. We further demonstrate that concomitant Myod and Igf2 inactivation accelerates differentiation of a brown preadipocyte cell line and induces lipid accumulation and increased Ucp1 and Prdm16 expression. This in vitro approach brings additional support for the implication of both Myod and Igf2 in BAT development. These results provide the first in vivo evidence that a myogenic regulator together with a growth factor act simultaneously but through independent pathways to repress Prdm16, which opens potential therapeutic perspectives for human metabolic disorders.

Muscle satellite cells are primed for myogenesis but maintain quiescence with sequestration of Myf5 mRNA targeted by microRNA-31 in mRNP granules.

Regeneration of adult tissues depends on stem cells that are primed to enter a differentiation program, while remaining quiescent. How these two characteristics can be reconciled is exemplified by skeletal muscle in which the majority of quiescent satellite cells transcribe the myogenic determination gene Myf5, without activating the myogenic program. We show that Myf5 mRNA, together with microRNA-31, which regulates its translation, is sequestered in mRNP granules present in the quiescent satellite cell. In activated satellite cells, mRNP granules are dissociated, relative levels of miR-31 are reduced, and Myf5 protein accumulates, which initially requires translation, but not transcription. Conditions that promote the continued presence of mRNP granules delay the onset of myogenesis. Manipulation of miR-31 levels affects satellite cell differentiation ex vivo and muscle regeneration in vivo. We therefore propose a model in which posttranscriptional mechanisms hold quiescent stem cells poised to enter a tissue-specific differentiation program.

H19 antisense RNA can up-regulate Igf2 transcription by activation of a novel promoter in mouse myoblasts.

It was recently shown that a long non-coding RNA (lncRNA), that we named the 91H RNA (i.e. antisense H19 transcript), is overexpressed in human breast tumours and contributes in trans to the expression of the Insulin-like Growth Factor 2 (IGF2) gene on the paternal chromosome. Our preliminary experiments suggested that an H19 antisense transcript having a similar function may also be conserved in the mouse. In the present work, we further characterise the mouse 91H RNA and, using a genetic complementation approach in H19 KO myoblast cells, we show that ectopic expression of the mouse 91H RNA can up-regulate Igf2 expression in trans despite almost complete unmethylation of the Imprinting-Control Region (ICR). We then demonstrate that this activation occurs at the transcriptional level by activation of a previously unknown Igf2 promoter which displays, in mouse tissues, a preferential mesodermic expression (Pm promoter). Finally, our experiments indicate that a large excess of the H19 transcript can counteract 91H-mediated Igf2 activation. Our work contributes, in conjunction with other recent findings, to open new horizons to our understanding of Igf2 gene regulation and functions of the 91H/H19 RNAs in normal and pathological conditions.

Characterization of Pax3-expressing cells from adult blood vessels.

We report expression of Pax3, an important regulator of skeletal muscle stem cell behaviour, in the brachial and femoral arteries of adult mice. In these contractile arteries of the limb, but not in the elastic arteries of the trunk, bands of GFP-positive cells were observed in Pax3(GFP/+) mice. Histological and biochemical examination of the vessels, together with clonal analysis after purification of Pax3-GFP-positive cells by flow cytometry, established their vascular smooth muscle identity. These blood-vessel-derived cells do not respond to inducers of other mesodermal cell types, such as bone, however, they can contribute to muscle fibre formation when co-cultured with skeletal muscle cells. This myogenic conversion depends on the expression of Pax3, but is rare and non-cell autonomous as it requires cell fusion. Myocardin, which promotes acquisition of a mature smooth muscle phenotype in these Pax3-GFP-positive cells, antagonises their potential for skeletal muscle differentiation. Genetic manipulation shows that myocardin is, however, positively regulated by Pax3, unlike genes for other myocardin-related factors, MRTFA, MRTFB or SRF. Expression of Pax3 overlaps with that reported for Msx2, which is required for smooth muscle differentiation of blood vessel-derived multipotent mesoangioblasts. These observations are discussed with respect to the origin and function of Pax3-expressing cells in blood vessels, and more general questions of cell fate determination and adult cell plasticity and reprogramming.

Sex, fiber-type, and age dependent in vitro proliferation of mouse muscle satellite cells.

During postnatal growth and after muscle injury, satellite cells proliferate and differentiate into myotubes to form and repair musculature. Comparison of studies on satellite cell proliferation and differentiation characteristics is confounded by the heterogeneity of the experimental conditions used. To examine the influence of sex, age, and fiber-type origin on in vitro properties of satellite cells derived from postnatal muscles, fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles were extracted from male and female mice of 1 week to 3 months of age. Myoblast proliferation and myogenic regulatory factor (MRF) expression was measured from cultures of freshly isolated satellite cells. Higher proliferation rate and elevated Myod1 expression was found in male EDL and SOL derived cells compared with females at age of 40, 60, and 120 days, whereas inverse tendency for cell proliferation was apparent in EDL of juvenile (7-day-old) pups. Myogenin and Mrf4 transcripts were generally elevated in males of 40 and 60 days of age and in female EDL of juveniles. However, these differentiation markers did not significantly correlate with proliferation rate at all ages. Pax7, whose overexpression can block myogenesis, was up-regulated especially in 40-day-old females where MRF expression was low. These results indicate that gender, postnatal age, and muscle fiber origin affect proliferation and muscle transcription factor expression in vitro. The results also support the view that satellite cells originating from slow and fast muscles are intrinsically different and warrant further studies on the effect of cell origin for therapeutic approaches.

An adult tissue-specific stem cell in its niche: a gene profiling analysis of in vivo quiescent and activated muscle satellite cells.

The satellite cell of skeletal muscle provides a paradigm for quiescent and activated tissue stem cell states. We have carried out transcriptome analyses on satellite cells purified by flow cytometry from Pax3(GFP/+) mice. We compared samples from adult skeletal muscles where satellite cells are mainly quiescent, with samples from growing muscles or regenerating (mdx) muscles, where they are activated. Analysis of regulation that is shared by both activated states avoids other effects due to immature or pathological conditions. This in vivo profile differs from that of previously analyzed satellite cells activated after cell culture. It reveals how the satellite cell protects itself from damage and maintains quiescence, while being primed for activation on receipt of the appropriate signal. This is illustrated by manipulation of the corepressor Dach1, and by the demonstration that quiescent satellite cells are better protected from oxidative stress than those from mdx or 1-week-old muscles. The quiescent versus in vivo activated comparison also gives new insights into how the satellite cell controls its niche on the muscle fiber through cell adhesion and matrix remodeling. The latter also potentiates growth factor activity through proteoglycan modification. Dismantling the extracellular matrix is important for satellite cell activation when the expression of proteinases is up-regulated, whereas transcripts for their inhibitors are high in quiescent cells. In keeping with this, we demonstrate that metalloproteinase function is required for efficient regeneration in vivo.