Sprouty1 Regulates Reversible Quiescence of a Self-Renewing Adult Muscle Stem Cell Pool during Regeneration

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Highlights? Pax7⁺ skeletal satellite cells regenerate muscle in their endogenous environment ? Quiescent satellite cells express the RTK inhibitor Spry1 ? Spry1 is needed for satellite cells to return to quiescence after muscle injury ? Deletion of Spry1 in satellite cells depletes the muscle stem cell pool     

Extraordinarily rapid proliferation of cultured muscle satellite cells from migratory birds

Migratory birds experience bouts of muscle growth and depletion as they prepare for, and undertake prolonged flight. Our studies of migratory bird muscle physiology in vitro led to the discovery that sanderling (Calidris alba) muscle satellite cells proliferate more rapidly than other normal cell lines. Here we determined the proliferation rate of muscle satellite cells isolated from five migratory species (sanderling; ruff, Calidris pugnax; western sandpiper, Calidris mauri; yellow-rumped warbler, Setophaga coronata; Swainson''s thrush, Catharus ustulatus) from two families (shorebirds and songbirds) and with different migratory strategies. Ruff and sanderling satellite cells exhibited rapid proliferation, with population doubling times of 9.3 ± 1.3 and 11.4 ± 2 h, whereas the remaining species'' cell doubling times were greater than or equal to 24 h. The results indicate that the rapid proliferation of satellite cells is not associated with total migration distance but may be related to flight bout duration and interact with lifespan.     

Sphingosine-1-phosphate receptor 3 influences cell cycle progression in muscle satellite cells

Skeletal muscle retains a resident stem cell population called satellite cells, which are mitotically quiescent in mature muscle, but can be activated to produce myoblast progeny for muscle homeostasis, hypertrophy and repair. We have previously shown that satellite cell activation is partially controlled by the bioactive phospholipid, sphingosine-1-phosphate, and that S1P biosynthesis is required for muscle regeneration. Here we investigate the role of sphingosine-1-phosphate receptor 3 (S1PR3) in regulating murine satellite cell function. S1PR3 levels were high in quiescent myogenic cells before falling during entry into cell cycle. Retrovirally-mediated constitutive expression of S1PR3 led to suppressed cell cycle progression in satellite cells, but did not overtly affect the myogenic program. Conversely, satellite cells isolated from S1PR3-null mice exhibited enhanced proliferation ex-vivo. In vivo, acute cardiotoxin-induced muscle regeneration was enhanced in S1PR3-null mice, with bigger muscle fibres compared to control mice. Importantly, genetically deleting S1PR3 in the mdx mouse model of Duchenne muscular dystrophy produced a less severe muscle dystrophic phenotype, than when signalling though S1PR3 was operational. In conclusion, signalling though S1PR3 suppresses cell cycle progression to regulate function in muscle satellite cells.     

Notch signaling deficiency underlies age-dependent depletion of satellite cells in muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating disease characterized by muscle wasting, loss of mobility and death in early adulthood. Satellite cells are muscle-resident stem cells responsible for the repair and regeneration of damaged muscles. One pathological feature of DMD is the progressive depletion of satellite cells, leading to the failure of muscle repair. Here, we attempted to explore the molecular mechanisms underlying satellite cell ablation in the dystrophin mutant mdx mouse, a well-established model for DMD. Initial muscle degeneration activates satellite cells, resulting in increased satellite cell number in young mdx mice. This is followed by rapid loss of satellite cells with age due to the reduced self-renewal ability of mdx satellite cells. In addition, satellite cell composition is altered even in young mdx mice, with significant reductions in the abundance of non-committed (Pax7⁺ and Myf5⁻) satellite cells. Using a Notch-reporter mouse, we found that the mdx satellite cells have reduced activation of Notch signaling, which has been shown to be necessary to maintain satellite cell quiescence and self-renewal. Concomitantly, the expression of Notch1, Notch3, Jag1, Hey1 and HeyL are reduced in the mdx primary myoblast. Finally, we established a mouse model to constitutively activate Notch signaling in satellite cells, and show that Notch activation is sufficient to rescue the self-renewal deficiencies of mdx satellite cells. These results demonstrate that Notch signaling is essential for maintaining the satellite cell pool and that its deficiency leads to depletion of satellite cells in DMD.KEY WORDS: Muscular dystrophy, Notch signaling, Stem cell     

Delineating the relationship between immune system aging and myogenesis in muscle repair

Recruited immune cells play a critical role in muscle repair, in part by interacting with local stem cell populations to regulate muscle regeneration. How aging affects their communication during myogenesis is unclear. Here, we investigate how aging impacts the cellular function of these two cell types after muscle injury during normal aging or after immune rejuvenation using a young to old (Y‐O) or old to old (O‐O) bone marrow (BM) transplant model. We found that skeletal muscle from old mice (20 months) exhibited elevated basal inflammation and possessed fewer satellite cells compared with young mice (3 months). After cardiotoxin muscle injury (CTX), old mice exhibited a blunted inflammatory response compared with young mice and enhanced M2 macrophage recruitment and IL‐10 expression. Temporal immune and cytokine responses of old mice were partially restored to a young phenotype following reconstitution with young cells (Y‐O chimeras). Improved immune responses in Y‐O chimeras were associated with greater satellite cell proliferation compared with O‐O chimeras. To identify how immune cell aging affects myoblast function, conditioned media (CM) from activated young or old macrophages was applied to cultured C2C12 myoblasts. CM from young macrophages inhibited myogenesis while CM from old macrophages reduced proliferation. These functional differences coincided with age‐related differences in macrophage cytokine expression. Together, this study examines the infiltration and proliferation of immune cells and satellite cells after injury in the context of aging and, using BM chimeras, demonstrates that young immune cells retain cell autonomy in an old host to increase satellite cell proliferation.     

Myogenin regulates a distinct genetic program in adult muscle stem cells

In contrast to the detailed understanding we have for the regulation of skeletal muscle gene expression in embryos, similar insights into postnatal muscle growth and regeneration are largely inferential or do not directly address gene regulatory mechanisms. Muscle stem cells (satellite cells) are chiefly responsible for providing new muscle during postnatal and adult life. The purpose of this study was to determine the role that the myogenic basic helix-loop-helix regulatory factor myogenin has in postnatal muscle growth and adult muscle stem cell gene expression. We found that myogenin is absolutely required for skeletal muscle development and survival until birth, but it is dispensable for postnatal life. However, Myog deletion after birth led to reduced body size implying a role for myogenin in regulating body homeostasis. Despite a lack of skeletal muscle defects in Myog-deleted mice during postnatal life and the efficient differentiation of cultured Myog-deleted adult muscle stem cells, the loss of myogenin profoundly altered the pattern of gene expression in cultured muscle stem cells and adult skeletal muscle. Remarkably, these changes in gene expression were distinct from those found in Myog-null embryonic skeletal muscle, indicating that myogenin has separate functions during postnatal life.     

Skeletal Muscle Satellite Cells Are Committed to Myogenesis and Do Not Spontaneously Adopt Nonmyogenic Fates

The developmental potential of skeletal muscle stem cells (satellite cells) remains controversial. The authors investigated satellite cell developmental potential in single fiber and clonal cultures derived from MyoD^iCre/+;R26R^EYFP/+ muscle, in which essentially all satellite cells are permanently labeled. Approximately 60% of the clones derived from cells that co-purified with muscle fibers spontaneously underwent adipogenic differentiation. These adipocytes stained with Oil-Red-O and expressed the terminal differentiation markers, adipsin and fatty acid binding protein 4, but did not express EYFP and were therefore not of satellite cell origin. Satellite cells mutant for either MyoD or Myf-5 also maintained myogenic programming in culture and did not adopt an adipogenic fate. Incorporation of additional wash steps prior to muscle fiber plating virtually eliminated the non-myogenic cells but did not reduce the number of adherent Pax7+ satellite cells. More than half of the adipocytes observed in cultures from Tie2-Cre mice were recombined, further demonstrating a non-satellite cell origin. Under adipogenesis-inducing conditions, satellite cells accumulated cytoplasmic lipid but maintained myogenic protein expression and did not fully execute the adipogenic differentiation program, distinguishing them from adipocytes observed in muscle fiber cultures. The authors conclude that skeletal muscle satellite cells are committed to myogenesis and do not spontaneously adopt an adipogenic fate.     

Satellite cells in mature,uninjured skeletal muscle of the lizard tail

The uninjured caudal skeletal muscle of two lizards, Lygosoma and Anolis, contains satellite cells. The satellite cell nuclei constitute 7.5% and 4.8% of the combined satellite and muscle nuclei, in Lygosoma and Anolis, respectively.     

Reduced proliferative activity of primary POMGnT1-null myoblasts in vitro

Protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) is an enzyme that transfers N-acetylglucosamine to O-mannose of glycoproteins. Mutations of the POMGnT1 gene cause muscle–eye–brain (MEB) disease. To obtain a better understanding of the pathogenesis of MEB disease, we mutated the POMGnT1 gene in mice using a targeting technique. The mutant muscle showed aberrant glycosylation of α-DG, and α-DG from mutant muscle failed to bind laminin in a binding assay. POMGnT1^?/? muscle showed minimal pathological changes with very low-serum creatine kinase levels, and had normally formed muscle basal lamina, but showed reduced muscle mass, reduced numbers of muscle fibers, and impaired muscle regeneration. Importantly, POMGnT1^?/? satellite cells proliferated slowly, but efficiently differentiated into multinuclear myotubes in vitro. Transfer of a retrovirus vector-mediated POMGnT1 gene into POMGnT1^?/? myoblasts completely restored the glycosylation of α-DG, but proliferation of the cells was not improved. Our results suggest that proper glycosylation of α-DG is important for maintenance of the proliferative activity of satellite cells in vivo.     

FOXP3+ T Cells Recruited to Sites of Sterile Skeletal Muscle Injury Regulate the Fate of Satellite Cells and Guide Effective Tissue Regeneration

Muscle injury induces a classical inflammatory response in which cells of the innate immune system rapidly invade the tissue. Macrophages are prominently involved in this response and required for proper healing, as they are known to be important for clearing cellular debris and supporting satellite cell differentiation. Here, we sought to assess the role of the adaptive immune system in muscle regeneration after acute damage. We show that T lymphocytes are transiently recruited into the muscle after damage and appear to exert a pro-myogenic effect on muscle repair. We observed a decrease in the cross-sectional area of regenerating myofibers after injury in Rag2^-/- γ-chain^-/- mice, as compared to WT controls, suggesting that T cell recruitment promotes muscle regeneration. Skeletal muscle infiltrating T lymphocytes were enriched in CD4⁺CD25⁺FOXP3⁺ cells. Direct exposure of muscle satellite cells to in vitro induced Treg cells effectively enhanced their expansion, and concurrently inhibited their myogenic differentiation. In vivo, the recruitment of Tregs to acutely injured muscle was limited to the time period of satellite expansion, with possibly important implications for situations in which inflammatory conditions persist, such as muscular dystrophies and inflammatory myopathies. We conclude that the adaptive immune system, in particular T regulatory cells, is critically involved in effective skeletal muscle regeneration. Thus, in addition to their well-established role as regulators of the immune/inflammatory response, T regulatory cells also regulate the activity of skeletal muscle precursor cells, and are instrumental for the proper regeneration of this tissue.     

Embryonic founders of adult muscle stem cells are primed by the determination gene Mrf4

Skeletal muscle satellite cells play a critical role during muscle growth, homoeostasis and regeneration. Selective induction of the muscle determination genes Myf5, Myod and Mrf4 during prenatal development can potentially impact on the reported functional heterogeneity of adult satellite cells. Accordingly, expression of Myf5 was reported to diminish the self-renewal potential of the majority of satellite cells. In contrast, virtually all adult satellite cells showed antecedence of Myod activity. Here we examine the priming of myogenic cells by Mrf4 throughout development. Using a Cre-lox based genetic strategy and novel highly sensitive Pax7 reporter alleles compared to the ubiquitous Rosa26-based reporters, we show that all adult satellite cells, independently of their anatomical location or embryonic origin, have been primed for Mrf4 expression. Given that Mrf4^Cre and Mrf4^nlacZ are active exclusively in progenitors during embryogenesis, whereas later expression is restricted to differentiated myogenic cells, our findings suggest that adult satellite cells emerge from embryonic founder cells in which the Mrf4 locus was activated. Therefore, this level of myogenic priming by induction of Mrf4, does not compromise the potential of the founder cells to assume an upstream muscle stem cell state. We propose that embryonic myogenic cells and the majority of adult muscle stem cells form a lineage continuum.     

A role for the myogenic determination gene Myf5 in adult regenerative myogenesis

The myogenic determination genes Myf5, Myod and Mrf4 direct skeletal muscle cell fate prenatally. In adult myogenesis, Myod has been shown to regulate myoblast differentiation, however, our understanding of satellite cell regulation is incomplete since the roles of Myf5 and Mrf4 had not been clearly defined. Here we examine the function of Myf5 and Mrf4 in the adult using recently generated alleles. Mrf4 is not expressed in normal or Myf5 null satellite cells and myoblasts, therefore excluding a role for this determination gene in adult muscle progenitors. Skeletal muscles of adult Myf5 null mice exhibit a subtle progressive myopathy. Crucially, adult Myf5 null mice exhibit perturbed muscle regeneration with a significant increase in muscle fibre hypertrophy, delayed differentiation, adipocyte accumulation, and fibrosis after freeze-injury. Satellite cell numbers are not significantly altered in Myf5 null animals and they show a modest impaired proliferation under some conditions in vitro. Mice double mutant for Myf5 and Dystrophin were more severely affected than single mutants, with enhanced necrosis and regeneration. Therefore, we show that Myf5 is a regulator of regenerative myogenesis and homeostasis, with functions distinct from those of Myod and Mrf4.     

Requirement of Plasminogen Binding to Its Cell-Surface Receptor α-Enolase for Efficient Regeneration of Normal and Dystrophic Skeletal Muscle

Adult regenerative myogenesis is central for restoring normal tissue structure and function after muscle damage. In muscle repair after injury, as in severe myopathies, damaged and necrotic fibers are removed by infiltrating inflammatory cells and then replaced by muscle stem cells or satellite cells, which will fuse to form new myofibers. Extracellular proteolysis mediated by uPA-generated plasmin plays a critical role in controlling inflammation and satellite-cell-dependent myogenesis. α-enolase has been described as plasminogen receptor in several cell types, where it acts concentrating plasmin proteolytic activity on the cell surface. In this study, we investigated whether α-enolase plasminogen receptor plays a regulatory role during the muscular repair process. Inhibitors of α-enolase/plasminogen binding: MAb11G1 (a monoclonal antibody against α-enolase) and ε-aminocaproic acid, EACA (a lysine analogue) inhibited the myogenic abilities of satellite cells-derived myoblasts. Furthermore, knockdown of α-enolase decreased myogenic fusion of myoblasts. Injured wild-type mice and dystrophic mdx mice were also treated with MAb11G1 and EACA. These treatments had negative impacts on muscle repair impairing satellite cell functions in vitro in agreement with blunted growth of new myofibers in vivo. Furthermore, both MAb11G1 and EACA treatments impaired adequate inflammatory cell infiltration and promoted extracellular matrix deposition in vivo, which resulted in persistent degeneration. These results demonstrate the novel requirement of α-enolase for restoring homeostasis of injured muscle tissue, by controlling the pericellular localization of plasmin activity.     

Reduced satellite cell density and myogenesis in Wagyu compared with Angus cattle as a possible explanation of its high marbling

Mechanisms responsible for excellent marbling in Japanese black cattle, Wagyu, remain to be established. Because both muscle cells and intramuscular adipocytes are developed from mesenchymal progenitor cells during early muscle development, we hypothesized that intramuscular progenitor cells in Wagyu cattle have attenuated myogenic capacity in favor of adipogenesis, leading to high marbling but reduced muscle growth. Biceps femoris muscle biopsy samples were obtained from both Angus (n=3) and Wagyu (n=3) cattle at 12 months of age. Compared with Angus, the density of satellite cells was much lower in Wagyu muscle (by 45.8±10%, P<0.05). Consistently, the formation of myotubes from muscle-derived progenitor cells was also lower (by 64.2±12.9%, P<0.05), but adipogenic capacity was greater in Wagyu. The average muscle fiber diameter was larger in Wagyu (by 23.9±6.8%, P=0.089) despite less muscle mass, suggesting less muscle fiber formation in Wagyu compared with Angus cattle. Because satellite cells are derived from fetal myogenic cells, the reduction in satellite cell density together with lower muscle fiber formation suggests that myogenesis was attenuated during early muscle development in Wagyu cattle. Given the shared pool of mesenchymal progenitor cells, the attenuated myogenesis likely shifts progenitor cells to adipogenesis during early development, which may contribute to high intramuscular adipocyte formation in Wagyu cattle.     

Progenitors of skeletal muscle satellite cells express the muscle determination gene, MyoD

Satellite cells are tissue-specific stem cells responsible for skeletal muscle growth and regeneration. Although satellite cells were identified almost 50 years ago, the identity of progenitor populations from which they derive remains controversial. We developed MyoD^iCre knockin mice, and used Cre/lox lineage analysis to determine whether satellite cell progenitors express MyoD, a marker of myogenic commitment. Recombination status of satellite cells was determined by confocal microscopy of isolated muscle fibers and by electron microscopic observation of muscle tissue fixed immediately following isolation, using R26R-EYFP and R26R (β-gal) reporter mice, respectively. We show that essentially all adult satellite cells associated with limb and body wall musculature, as well as the diaphragm and extraocular muscles, originate from MyoD+ progenitors. Neonatal satellite cells were Cre-recombined, but only a small minority exhibited ongoing Cre expression, indicating that most satellite cells had expressed MyoD prenatally. We also show that satellite cell development in MyoD-null mice is not due to functional compensation by MyoD non-expressing lineages. The results suggest that satellite cells are derived from committed myogenic progenitors, irrespective of the anatomical location, embryological origin, or physiological properties of associated musculature.     

Six family genes control the proliferation and differentiation of muscle satellite cells

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4^+/−Six5^−/− mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.