Satellite Cells and Skeletal Muscle Regeneration.

Satellite Cells and Skeletal Muscle Regeneration Nicolas A. Dumont,1 C. Florian Bentzinger,1,2 Marie-Claude Sincennes,1 and Michael A. Rudnicki*1,3 ABSTRACT Skeletal muscles are essential for vital functions such as movement, postural support, breathing, and thermogenesis. Muscle tissue is largely composed of long, postmitotic multinucleated fibers. The life-long maintenance of muscle tissue is mediated by satellite cells, lying in close proximity to the muscle fibers. Muscle satellite cells are a heterogeneous population with a small subset of muscle stem cells, termed satellite stem cells. Under homeostatic conditions all satellite cells are poised for activation by stimuli such as physical trauma or growth signals. After activation, satellite stem cells undergo symmetric divisions to expand their number or asymmetric divisions to give rise to cohorts of committed satellite cells and thus progenitors. Myogenic progenitors proliferate, and eventually differentiate through fusion with each other or to damaged fibers to reconstitute fiber integrity and function. In the recent years, research has begun to unravel the intrinsic and extrinsic mechanisms controlling satellite cell behavior. Nonetheless, an understanding of the complex cellular and molecular interactions of satellite cells with their dynamic microenvironment remains a major challenge, especially in pathological conditions. The goal of this review is to comprehensively summarize the current knowledge on satellite cell characteristics, functions, and behavior in muscle regeneration and in pathological conditions. © 2015 American Physiological Society. Compr Physiol 5:1027-1059, 2015.

Introduction Striated skeletal muscle is the most abundant tissue of the human body, representing approximately 35% to 45% of total body mass (238). In contrast to heart muscle, skeletal muscle is controlled by the somatic nervous system that enables voluntary contraction. There are more than 600 skeletal muscles of different sizes and contractile properties that allow for various functions such as locomotion, postural support, powerful or precise movements, and breathing. One of the most remarkable capacities of skeletal muscle is its very high adaptive potential. For example, resistance training is well known to induce muscle hypertrophy and increase muscle strength. In contrast, many different diseases or conditions such as cancer, chronic obstructive pulmonary diseases, heart failure, aging, denervation, or disuse can lead to muscle atrophy and loss of function (10, 98). Next to their high plasticity, skeletal muscles also possess exceptional regenerative capacity. Only few weeks after a major injury destroying fiber integrity, skeletal muscle structure and function can be completely restored (24). The mediators of muscle regeneration are a population of small adult stem cells, termed satellite cells, located in close proximity to muscle fibers. Under resting conditions, adult satellite cells are quiescent, but they can quickly re-enter the cell cycle following injuries or growth signals. Activated satellite cells will migrate extensively, proliferate, differentiate, and fuse to form regenerating myofibers. Only a subset of self-renewing cells is able to withstand differentiation during muscle injury and will return to quiescence after completion of muscle regeneration to replenish the satellite cell pool and to prepare the tissue for a subsequent injury. Importantly, the fate of satellite

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cells is greatly influenced by intrinsic and extrinsic factors. Dynamic interactions with inflammatory cells, stromal cells, trophic signals, and extracellular matrix (ECM) components guide satellite cells through the regeneration process. However, many pathological conditions, such as muscular dystrophies (MDs) or muscle wasting, provide inadequate cues to the satellite cells and thereby impair their regenerative potential. This review will first describe satellite cell characteristics and their developmental origin. Subsequently, satellite cell behavior during the different steps of muscle regeneration will be discussed and will be followed by a review of the intrinsic and extrinsic factors regulating satellite cell fate during adult myogenesis. Finally, the effects of diverse degenerative conditions on satellite cells, as well as the different therapeutic avenues will be summarized.

Historical Perspective It has been known since the 19th century that muscle tissue possesses great regenerative potential even following serious traumas such as ischemia, burns, incisions, or crush injuries * Correspondence

to [email protected] Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada 2 Nestl´ e Institute of Health Sciences, EPFL Campus, Lausanne, Switzerland 3 Faculty of Medicine, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada Published online, July 2015 (comprehensivephysiology.com) DOI: 10.1002/cphy.c140068 Copyright © American Physiological Society. 1 Sprott

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(264). Initially, the basic characteristics of muscle regeneration such as leukocyte infiltration, fibrotic tissue formation, revascularization, and repair of damaged fibers were characterized (64). However, the origin of de novo myofiber formation was unclear and various hypotheses were proposed, for instance, the splitting of intact myofibers, the elongation of damaged myofibers, the formation of new myofibers from circulating leukocytes, or the proliferation of myonuclei. The first evidence for the presence of muscle resident cells with myogenic potential was revealed by an in vitro study showing that culture of small pieces of chick embryonic muscles results in expansion of a myoblast population and formation of multinucleated myotubes (175). Alexander Mauro was the first to identify in vivo a population of small mononuclear cells that he named satellite cells based on their location between the plasma membrane of the myofiber and the basement membrane (192). First described in frogs, these satellite cells were also observed in skeletal muscles from various mammalian species, including humans (140). Mauro suggested that satellite cells are a dormant cell type that could be activated to promote regeneration upon fiber damage. The development of radioactive nucleoside labeling techniques using tritiated thymidine demonstrated that only mononuclear cells, and not myonuclei, were actively synthesizing new DNA strands (29). However, a short radioactive pulse followed by muscle injury showed that a radioactive signal could be observed in the nuclei of newly regenerated myotubes, which suggested that activated satellite cells are able to fuse to damaged myofibers. The fusion ability of myoblasts was confirmed using allophenic mouse models, where hybrid fibers formed from the fusion of cells with different genetic backgrounds were observed (45, 202). Subsequently, single fiber isolation techniques were useful to

Osteotendinous junction

Comprehensive Physiology

confirm that satellite cells lying underneath the basal lamina are the cell type responsible for myoblast proliferation and myotube formation (31). The discovery of myogenic regulatory factors (MRFs) and paired box proteins then allowed to further characterize myogenic cell behavior during the different steps of myogenesis (102, 120, 198, 269, 316). More recently, another important hallmark of satellite cell behavior was discovered. Using transplantation experiments in radiation-ablated muscles, it was shown that freshly isolated satellite cells possess a high self-renewal capacity (67, 163, 255). These results were also accompanied by the discovery of considerable heterogeneity within the satellite cell population. Satellite cells can, for instance, be subdivided into a subpopulation of satellite cells that are already committed to become myogenic progenitors and a subpopulation of satellite stem cells with higher self-renewal capacity (163). The latter population was also shown to be able to perform asymmetric division and give rise to committed satellite cells (163). Thus, the ability of a subset of satellite cells to self-renew and to perform asymmetric division confirmed these cells as adult stem cells. The following sections of this review will describe our current understanding of satellite cell biology.

Anatomy Skeletal muscles are mostly composed of long parallel cylindrical fibers attached together by a complex and structured ECM (Fig. 1). Each fiber is enveloped by layers of ECM, namely, the endomysium and the basement membrane (188). The endomysium is the most exterior membrane and is mainly composed of collagen fibrils interlaced with a vast network of Myotendinous junction

Skeletal muscle

Bone Basal lamina

Joint

Satellite Blood cell vessel

Multinucleated muscle fiber

Figure 1 Skeletal muscle anatomy. Schematic illustrating the structure of skeletal muscle. Skeletal muscles are divided into bundles containing numerous myofibers. Myofibers are multinucleated cylindrical cells that are surrounded by a vast network of blood vessels and nerves. Satellite cells are small mononuclear cells located between the plasmalemma of the myofibers and the basal membrane.

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Figure 2 Satellite cell markers. Images of single extensor digitorium longus (EDL) myofiber labeled for different markers of satellite cells. The left image shows a multinucleated myofiber with two satellite cells stained by Pax7 antibody (red) and a nuclear dye (DAPI, blue). The right picture displays a satellite cell stained with M-cadherin (red) on the apical side and α7-integrin (white) on the basal side.

blood vessels and nerves. Lying between the endomysium and the fiber membrane (plasmalemma), the basement membrane can be further divided into the reticular lamina (collagen fibrils) and the basal lamina (collagen IV, fibronectin, laminin, and proteoglycans) (167, 262). The plasmalemma contains numerous proteins such as the dystroglycan-sarcoglycandystrophin complex that are involved in linking the fiber to the ECM. Skeletal muscle fibers are distinct from every other cell type of the body. Their interior is mainly constituted of myofibrils, which contain actin and myosin filaments responsible for muscle contraction. Muscle fibers are very long cells that can measure up to 20 to 30 cm in humans (131). To fulfill the high-protein synthesis demand of these long contractile cells, muscle fibers possess numerous myonuclei. Myonuclei cover a relatively constant territory ranging from 2 × 104 to 5 × 104 μm3 , although the existence of a defined nuclear domain is controversial (121, 178, 193). Importantly, myonuclei are postmitotic and are not able to divide. The satellite cells that populate these fibers are wedged between the basal lamina and the plasmalemma and more than 80% to 90% of them are located only a few micrometers away from a blood vessel (Fig. 1) (61). Satellite cells constitute 2% to 10% of total myonuclei, which represents approximately 2 × 105 to 1 × 106 satellite cells per gram of muscle (25,128). However, the proportion of satellite cell depends on many factors such as muscle type, age, and species. For example, slow oxidative muscles contain more satellite cells than fast glycolytic muscles, probably to satisfy the repair demand of these highly active postural muscles (109).

Satellite Cell Characteristics Apart from their anatomic location, satellite cells can also be distinguished based on their cellular characteristics, for instance, their high nucleus-to-cytoplasm ratio. In addition,

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many different proteins were discovered as markers of satellite cells. Some markers are located in the nucleus such as the paired box proteins PAX3 and PAX7 (Fig. 2). In adult muscles, Pax7 is highly and constantly expressed in satellite cells, while Pax3 is usually expressed at very low levels except in a subset of muscles such as the diaphragm (152,206). Other transcription factors such as myogenic factor 5 (Myf5) and myogenic differentiation 1 (MyoD) can be specifically expressed by myogenic cells, but are not expressed in quiescent satellite cells. Finally, several cell surface membrane proteins were identified as markers of satellite cells such as, M-cadherin, α7- and β1-integrins, c-Met, C-X-C chemokine receptor type 4 (CXCR4), syndecan-3 and -4, calcitonin receptor, calveolin-1, CD34, vascular cell adhesion molecule-1 (VCAM1), and neural cell adhesion molecule-1 (NCAM1) (Fig. 2) (104, 113). Histologically, anti-laminin combined with anti-Mcadherin immunostaining allows for the identification of satellite cells in their niche (Fig. 3). However, Pax7 is the most widely recognized intracellular marker because it is ubiquitously expressed in all satellite cells in a wide variety of species. It is important to keep in mind that Pax7 is also expressed in activated satellite cells and is thus not a specific marker for quiescent satellite cells. Transgenic mice carrying Pax7 promoter-driven fluorochrome expression, such as Pax7zsgreen mice or tamoxifen-inducible fluorochrome expression in Pax7creER -YFP (yellow fluorescent protein) mice are very convenient research tools to isolate and/or analyze satellite cell behavior (23, 37). However, without these reporter mouse models Pax7 is an unpractical marker for viable satellite cell isolation by FACS due to its nuclear location. For cell sorting, positive selection for the satellite cell surface markers α7-integrin and CD34 combined with negative selection for markers of other cell types CD45 (hematopoietic lineage), CD11b (leukocytes), CD31 (endothelial cells and leukocytes), and Sca-1 (mesenchymal and hematopoietic stem cells) has been shown to isolate a pure population of satellite

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Figure 3

The satellite cell niche. Cross-section of a tibialis anterior (TA) muscle immunostained with laminin (white), M-cadherin (green), Pax7 (red), and a nuclear dye (DAPI, blue). All muscle fibers are surrounded by laminin. The insert in the top right corner shows a magnification of a satellite cell (Pax7-positive) in its niche.

cells from skeletal muscles (229). A monoclonal antibody, SM/C-2.6, was also developed to bind specifically to satellite cells although its antigen is still unknown (103).

Developmental Origin of Satellite Cells Three germ layers are formed during mammal embryogenesis: the ectoderm, mesoderm, and endoderm. The mesoderm can be further subdivided into lateral, intermediate, and paraxial mesoderm. Bilateral paired blocks, named somites, form from the paraxial mesoderm and develop along the anteriorposterior axis (head to tail) of the embryo (Fig. 4) (25). Somites give rise to a variety of tissues such as vertebral bones, tendons, cartilage, dermis, and skeletal muscles. All skeletal muscles, except for some head muscles, are derived from the dorsal portion of the somites, named the dermomyotome. Dorsal muscles arise from the epaxial part of the dermomyotome (adjacent to the neural tube), whereas limb muscles originate from the hypaxial part of the dermomyotome. A vast network of transcription factors controls myogenic cell specification, determination and differentiation (Fig. 5). Paired box domain transcription factors PAX3 and/or PAX7 are expressed by dermomyotome cells and are important upstream regulators of muscle tissue formation. Pax3 is critical during muscle development especially for the formation of hypaxial domain. Pax3 loss-of-function leads to absence of diaphragm and limb muscles, but this effect is less severe on epaxial-derived muscles (35, 301). Pax3 has been demonstrated to target the hepatocyte growth factor (HGF) receptor c-met, which is essential for the delamination and migration of the myogenic progenitors (96). Moreover,

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Figure 4

Embryonic muscle development. The image shows a mouse embryo transgenic for a fluorescent expression reporter (TdTomato) of the myogenic transcription factor Myf5 at 10.5 days after fertilization. At this developmental stage Myf5 is highly expressed in the somites. Somites develop antero-posteriorly, with the anterior somites being the most mature ones and the posterior somites the least developed.

Pax3 has been shown to regulate the expression of Myf5 and MyoD in the embryos, which triggers myogenic determination (239). Despite its essential role in muscle formation, absence of Pax3 in postnatal muscles does not impair adult muscle regeneration (245, 290). On the other hand, Pax7 deletion is much less severe for muscle development in the embryo, but is absolutely essential for satellite cell formation (162, 221, 244, 269). In adult myogenesis, the transcriptional

Pax3/7 + MRF–

Myf5 Pax3/7

Myogenin MRF4

MyoD Myogenic specification

Myogenic determination

Myogenic differentiation

Figure 5

Genetic programme regulating skeletal muscle stem cell fate. Hierarchical network of the transcription factors controlling embryonic progenitor cell fate during limb and trunk muscle development. Pax3 and Pax7 control the myogenic specification of embryonic progenitors. Pax3/7 can directly target Myf5 and MyoD expression and thus myogenic determination. Myf5 also acts partially upstream of MyoD and can trigger its expression. Upregulation of myogenin and MRF4 induces myogenic differentiation. A population of Pax3/7+ progenitor cells does not express MRFs and gives rise to satellite stem cells during late fetal myogenesis.

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dominance of Pax7 over Pax3 is explained by its higher binding affinity to the homeodomain binding motif (283). Pax7-null mice show gradual loss of satellite cells, reduced fiber size, and impaired muscle regeneration resulting in early postnatal death (122, 309). Lineage tracing experiments confirmed that Pax3-positive cells are essential for embryonic myogenesis, while Pax7-positive cells are more important for late fetal and postnatal myogenesis (137). Thus, it is hypothesized that Pax3-positive founder cells form a template of initial fibers, while Pax7-positive cells form secondary fibers and generate the satellite cell population (25, 30). Progression of embryonic progenitors (Pax3/7+ cells) through the myogenic program is regulated by different MRFs. MRFs are a family of four basic helix-loop-helix transcription factors: Myf5, MyoD, myogenin, and myogenic regulatory factor 4 (MRF4). Myf5 is the first MRF to be expressed, initially in the epaxial domain and subsequently in the hypaxial domain of the dermomyotome (42). MyoD is expressed after Myf5 in the hypaxial and epaxial domains. Myf5 and MyoD are critical factors for myogenic cell determination. Knockout of Myf5 or MyoD alone have a relatively mild effect on muscle development due to the functional overlap between these two factors (41, 251, 252). However, Myf5:MyoD double knockout mice show complete lack of skeletal muscle formation (252). Moreover, Myf5:MyoD double knockout mice do not express myogenin, which demonstrates the hierarchal relationship among the MRFs. Myogenin is essential for the terminal differentiation of myogenic cells and myogenin-null mice quickly die after birth from severe and global muscle deficiency (127). Similarly, MRF4null mice display myogenin overexpression, which suggests a role for this factor in late differentiation (242, 331). It has been suggested that MRF4 could also play a partial role as a determination factor in absence of Myf5 and MyoD; however, it has not been established whether it does the same in presence of Myf5 and MyoD (151). Globally, Myf5 and MyoD are determination factors required to establish myogenic identity, and act upstream of the differentiation factors myogenin and MRF4, but the exact hierarchical organization of the different MRFs is still a matter of debate (Fig. 5). While most Pax3/7+ cells express MRFs and commit to myogenic progenitors during development, there is a subset of Pax3/7+ cells that proliferates without upregulating the different MRFs (152, 246). While this might be true at least for the expression of Myf5, different reporter mice demonstrated that virtually all satellite cells transiently express MyoD prenatally (149). Nonetheless, it has been suggest that late in fetal development Pax3/7+MRF− cells align to the nascent myotubes and eventually adopt the satellite cell position under the basal lamina in postnatal muscles (Fig. 5) (246). Upon arrival to nascent muscles, the majority of Pax3/7+MRF− cells will downregulate Pax3 and quickly upregulate Myf5 (152). The presence of Pax7+Myf5- satellite stem cells in adult muscle might correspond to a lineage continuum of the Pax3/7+MRF− progenitors.

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Overall, there are many similarities between embryonic and adult myogenesis and many common transcription factors and signaling pathways are involved. Nevertheless, there are also many differences, especially in the microenvironment of the cells and in their level of activity.

Satellite Cell Heterogeneity Originally considered as a homogeneous population, different satellite cell subpopulations have recently been characterized. Some evidence suggests that there is a heterogeneity in satellite cells residing in slow versus fast muscles, for example, that satellite cells from slow muscles give rise to myofibers expressing slow myosin isoforms while satellite cells from fast muscles tend to form fast myosin (17, 97, 135, 148, 250). However, there is no clear data demonstrating intrinsic differences of satellite cells based on fiber type, particularly not in humans (36, 95). Expression of different markers also varies considerably between satellite cells. Paired box protein homologs Pax3 and Pax7 expression show a discrepancy depending on the muscle. For instance, more than 80% of satellite cells in diaphragm coexpress Pax3 and Pax7, while Pax3-expressing satellite cells are not found in hindlimb muscles (162). Expression of other satellite cell markers such as CD34, Myf5, and Mcadherin also vary among the satellite cell population (19). These markers are almost always coexpressed together in quiescent satellite cells, but they account only to approximately 80% of the total satellite cell population. Subpopulations of CD34neg , Myf5neg , and/or M-cadherinneg satellite cells were discovered and it was speculated that these cells could represent uncommitted progenitors responsible for the maintenance of the satellite cell population. Notably, the proportion of CD34neg and M-cadherinneg satellite cells decreases during aging (237). The presence of a satellite stem cell population within the satellite cell pool has been confirmed using Myf5-cre/R26R-YFP mice in which Myf5cre expression leads to permanent labeling with YFP. Using this mouse line it has been shown that approximately 10% of total satellite cells have never expressed Myf5 during development or postnatally (163). Transplantation experiments showed that Myf5neg satellite cells possess higher selfrenewal and engraftment capacity and are less prone to precocious differentiation than Myf5-positive (Myf5pos ) satellite cells. Myf5neg satellite cells are also able to perform asymmetric divisions and give rise to Myf5pos satellite cells (163). Myf5neg satellite cells are also relatively slow dividing relative to Myf5pos cells. These results confirm that there is heterogeneity within the satellite cell population, with a subpopulation of committed satellite cells (Myf5pos ) and a subpopulation of satellite stem cells (Myf5neg ). Another transgenic mouse model showed that satellite cells could be subdivided based on their level of Pax7 expression. Pax7Hi cells possess lower metabolic rate and slower

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division ability than Pax7Lo satellite cells that rather tend to express higher levels of myogenin (247). Consistently, labeling of satellite cells with the proliferation markers PKH26 or BrDU showed that most activated satellite cells undergo fast division, but a minority are slow-dividing (219,266,277). This observation is supported by a study using label retention as marker of satellite cell proliferative history throughout animal lifespan (53). In this study, it was shown that a subpopulation of satellite cells divides less frequently and retains the labeling (label-retaining cells) while another subpopulation loses the labeling over time (nonlabel retaining cells). Label retention experiments also demonstrated that slow-dividing cells can generate distinct daughter cells that are more prone to differentiation (52). However, the immortal strand hypothesis suggesting that one daughter stem cell retains the template DNA while the more committed cell is composed of the newly synthesized DNA strand, is still debated and no clear mechanism describing this phenomenon has yet been demonstrated (166). Taken together, the idea that satellite cells are heterogeneous and can be divided at least into a committed progenitor and a muscle stem cell subpopulation is experimentally well supported.

Satellite Cells in Muscle Regeneration Skeletal muscles are able to quickly regenerate mature myofibers following massive injuries that completely destroy muscle structure and integrity (Fig. 6). Deletion models confirmed that this remarkable regenerative capacity relies on the myogenic potential of satellite cells. Pax7-null mice have small myofibers at birth and are not able to form a functional satellite cell pool, which leads to rapid postnatal death (within few weeks of life) (162, 269). Conditional knockout models were more helpful to delineate the role of satellite cells in adult muscle regeneration. Pax7 depletion in adult mice using inducible Pax7flox/CreERT2 tamoxifen-treated mice strongly impaired the regenerative potential of skeletal muscle, indicating that Pax7 is also essential for the normal function of postnatal satellite cells (122, 309). In agreement with these findings, conditional depletion of satellite cells using mice Uninjured

2d

carrying the diphtheria toxin A gene under the control of the Pax7 locus showed that absence of satellite cells in injured muscles results in strong decrease in myofiber number and size, accompanied by massive fibrosis and intracellular fat accumulation (172,209,259). The deleterious effects of satellite cell ablation could be rescued by satellite cell transplantation into the depleted muscles (259). Intriguingly, satellite cell depletion completely blunts the regeneration of damaged muscles but it does not significantly impair the short-term hypertrophic capacity of muscle fibers in the absence of injury (194). Mechanical overload induced similar increases in muscle fiber cross sectional area and muscle force in presence or absence of satellite cells over 2 weeks. These results suggest that muscle fibers possess adaptive potential, that is, in part independent of satellite cells. However, the increase in myonuclei number usually associated with fiber hypertrophy is not observed in satellite celldeficient muscles, suggesting that the hypertrophic process is perturbed and that the myofibers are probably less functional. Moreover, the model of satellite cell depletion used by the authors (conditional depletion using diphtheria toxin A gene under the control of the Pax7 locus) leads to 90% depletion, leaving the possibility that the remaining satellite cells could contribute to the hypertrophic process (194). Muscle regeneration can be divided in several stages that are characterized by the expression of different MRFs (Fig. 7). In the quiescent stage satellite cells are inactive but are poised to activation. Quiescent satellite cells generally express markers such as Pax7 and Myf5 (except for the Myf5neg satellite stem cell population). After muscle injury, satellite cells are stimulated by the various signals arising from the damaged environment. Satellite cells then migrate toward the injury site and re-enter the cell cycle to proliferate. At this stage, committed satellite cells are termed myoblasts and express the myogenic markers Pax7, and/or Myf5, and/or MyoD (Fig. 8). After the proliferative phase, myoblasts exit the cell cycle and differentiate into mature myocytes. This differentiation process is accompanied by a decreased expression of Pax7 and Myf5, and by an increase in myogenin (MyoG) and MRF4 levels (Fig. 9). Finally, myocytes fuse together to form multinucleated myotubes and/or fuse to damaged myofibers. 5d

2mo

Figure 6 Skeletal muscle regeneration. H&E staining (nuclei stained in dark blue and cytosolic proteins stained in red) illustrating the different stages of muscle regeneration following cardiotoxin injury. Two days following injury, myofiber structure is severely impaired and a multitude of mononuclear cells (myoblasts, inflammatory cells, fibroblasts, etc.) are present in the damaged area. At five days postinjury, there is the formation of small new myofibers with centralized nuclei and many cells remain in the exudate. Two months after the injury, muscle architecture is similar to uninjured muscle.

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Embryonic progenitors


Satellite stem cells Pax7

Satellite cells Pax7 Myf5

Myoblasts Pax7 Myf5 MyoD

Myocytes MyoD Myogenin MRF4

Myotubes Myogenin MRF4 MyHC

Selfrenewal

Figure 7 Myogenic lineage progression. Following muscle injury quiescent satellite cells (Pax7+ and Myf5+/−) are activated and differentiate to myoblasts (Pax7+, Myf5+, and MyoD+). After several rounds of proliferation, myoblasts exit the cell cycle and become myocytes (Pax7−, MyoD+, myogenin+, and MRF4+). Myocytes can undergo a fusion process to form multinucleated myotubes (myogenin+, MRF4+, and MyHC+) that eventually mature into myofibers. The satellite stem cell subpopulation (Pax7+ and Myf5−) can also self-renew to replenish the satellite cell pool.

Figure 8

Myogenic regulatory factors in proliferating myoblasts. Images from single EDL myofibers cultured ex vivo for 60 h (dashed line delineates the myofiber membrane). Staining for Pax7 (green) and MyoD (red) shows that at this stage cells express either only Pax7 (arrowhead), only MyoD, or both (arrow).

At this stage, MyoD levels become reduced while myosin heavy chain (MyHC) and other contractile proteins start to be expressed. The balance between the different MRFs is critical to control cell fate. It was suggested that a high Pax7:MyoD ratio favors quiescence, an intermediate ratio enables proliferation but not differentiation, while a low Pax7:MyoD ratio promotes differentiation (218). During the myogenic lineage progression, satellite cell fate is influenced by a complex balance between intrinsic factors and external cues (24).

Satellite Cell Quiescence In adult resting muscles, satellite cells are in a quiescent state characterized by the absence of cell cycling (G0 phase), a low rate of metabolism and low RNA content (60). Genome-wide gene expression analysis comparing quiescent to activated satellite cells revealed more than 500 genes upregulated in quiescent satellite cells, which suggests that maintaining quiescence is an active process (104). These genes are mainly

Figure 9

Myogenic regulatory factors in differentiating myoblasts. Pictures from EDL isolated myofibers cultured ex vivo for 72 h (dashed line delineates the myofiber membrane). Staining for Pax7 (green) and myogenin (red) shows that cells that acquire the differentiation marker myogenin lose expression of Pax7.

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related to cell-cell interactions, formation of ECM, and regulation of cell growth. Some proteins such as Odz4 and the calcitonin receptor are almost exclusively expressed in dormant satellite cells and not expressed in activated satellite cells (325). Cell-cycle regulators, such as the retinoblastoma tumor suppressor protein Rb, are also highly enriched in quiescent satellite cells (104). In proliferating myoblasts, Rb protein is phosphorylated leading to its inactivation, while its dephosphorylation during terminal myogenic differentiation induces withdrawal from the cell cycle (71). Tamoxifen inducible ablation of Rb1 under the control of a Pax7 promoter leads to activation of quiescent satellite cells (134). Conditional Rb1 knockout triggers an expansion of the satellite cell population but also decreases the regenerative capacity of muscle due to impaired differentiation. Consistently, regulators of Rb protein, the cyclin-dependent kinase inhibitors (CKIs), are upregulated in quiescent satellite cells compared to activated satellite cells (104). Furthermore, the cell-cycle inhibitor p27kip1 is highly expressed in quiescent satellite cell (especially in the stem cell subpopulation) and knockout of this factor promotes proliferation and differentiation of satellite cells (52). Overall, there are many negative regulators that prevent cell cycle entry in quiescent satellite cells. The position of the satellite cells between the basal lamina and the fiber membrane suggests that the microenvironment of the cells could be involved in their regulation (205). For example, M-cadherin proteins present on both the satellite cell and the myofiber membrane interact together and “immobilize” the satellite cell (139). Adhesion molecules such as α7- and β1-integrins also interact with the basal lamina to stabilize the satellite cell in its niche. Moreover, quiescent satellite cells express high levels of tissue inhibitor of metalloproteinases (TIMPs) to block ECM degradation by metalloproteinases (MMPs) and therefore favor the integrity of the niche (223). A key signaling system involved in the control of satellite cell quiescence is the Notch pathway. The Notch ligand Delta1 is expressed at the fiber membrane and it appears likely that it interacts with Notch receptors present on the satellite cell membrane (70). In agreement with this idea, the Notch pathway is active in quiescent satellite cells. The cooperation of Notch receptor with Syndecan-3 is important to facilitate Notch signal transduction (233). Notch signaling activates the transcription factor Recombining binding protein suppressor of hairless (RBPJ) that triggers the activation of multiple genes such as Hes1, Hes5, Hey, and HeyL (319). Hey1 and HeyL double-knockout results in cell cycle re-entry of satellite cells and stimulates MyoD and myogenin expression (105). Similarly, Syndecan-3 knockout satellite cells display impaired Notch signaling leading to satellite cell exit from quiescence and lack of self-renewal (233). Thus, inhibition of the Notch pathway leads to activation and differentiation of satellite cells that ultimately results in the depletion of the satellite cell pool (33, 163, 208). On the other hand, overexpression of the constitutively active Notch intracellular domain (NICD)

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Table 1

Pathways Involved in the Different Myogenic Stages

Myogenic stage

Pathways

References

Quiescent satellite cells

Notch

Conboy 2002, Fukada 2011, Bjornson 2012, Mourikis 2012, Wen 2012 Gopinath 2014 Shea 2010, Abou-Khalil 2009

FOXO3 ERK1/2 (Sprouty1) Satellite cell activation

P38α/β MAPK Calcium (NFATc) Akt/mTOR NF-κB

Jones 2006 Liu 2014 Machida 2003 Acharyya 2010

Myogenic cell proliferation

JAK/STAT

Serrano 2008, Sun 2007 Otis 1994, Guttridge 1999 Perdigureo 2007, Alter 2008 Gillepsie 2009

NF-κB JNK P38γMAPK Myogenic differentiation/ fusion

Canonical Wnt p38αMAPK Calcium (calcineurin/ CaMK)

Myofiber hypertrophy

Akt/mTOR Myostatin

Self-renewal

Noncanonical Wnt (planar cell polarity) Notch

Brack 2008 Wu 2000, Perdiguero 2007, Lluis 2005 Friday 2003, Friday 2000, Lu 2000 Park 2005, Von Maltzahn 2012 McPherron 1997, Bogdanovich 2002 Legrand 2009, Bentzinger 2013 Gopinath 2014, Wen 2012, Kuang 2007

This table summarizes the major pathways involved in the different myogenic stages; satellite cell quiescence, activation, proliferation, differentiation/fusion, fiber hypertrophy, and self-renewal.

downregulates MyoD expression, reduces entry in S phase and decreases proliferation (319). In contrast to signals that actively maintain the dormant state, pathways involved in proliferation must be repressed in quiescent satellite cells (Table 1). Fibroblast growth factor-2 (FGF2) is an important signaling molecule involved in satellite cell activation (327). FGF2 signaling can be repressed by Sprouty-1, a receptor tyrosine kinase inhibitor. Sprouty-1 is highly enriched in quiescent satellite cells, is down-regulated in activated satellite cells, but is re-expressed when satellite cells return to quiescence. Sprouty-1-null mice display overactivated ERK1/2 pathway that lead to the failure of satellite cells to return to quiescence (274). Other signaling transduction inhibitors are enriched in quiescent satellite cells. For instance, Insulin growth factor binding protein-6 (Igfbp-6) is highly expressed by quiescent satellite cells and potentially antagonizes IGF signaling (223). It has been shown that quiescent satellite cells express Tie-2, a receptor that can interact with angiopoietin-1 (Ang1) secreted in part by vascular smooth muscle cells. Blocking Tie-2 results in increased numbers of cycling satellite cells,

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while overexpression of Ang-1 increases the number of quiescent satellite cells (2). Ang-1/Tie-2 signals through ERK1/2 to promote the G0 state and decrease proliferation and differentiation. These results suggest that neighboring vascular cells can help maintain satellite cell quiescence. MicroRNAs have been suggested to play a key role for the regulation of satellite cell quiescence. These small noncoding RNA molecules generally bind mRNA strands and posttranscriptionally regulate their translation. Hundreds of microRNAs were discovered so far, but a role for only a few of them have been described in muscle. Importantly, ablation of Dicer, the processing enzyme responsible for pre-microRNA hairpin cleavage and activation, leads to satellite cell exit of quiescence and entry into the cell cycle (59). By comparing microRNA expression of quiescent versus activated satellite cells, it was shown that 22 microRNAs are highly expressed in quiescent satellite cells and strongly downregulated following activation (59). The effects of some of these microRNAs were partially elucidated. For instance, miR-489 targets the oncogenic protein Dek that is involved in myogenic commitment and expansion of the myogenic progenitors (59). Another microRNA, miR-31, was shown to directly target Myf5 mRNA (75). Importantly, the myogenic determination factor Myf5 is expressed at the mRNA level in quiescent satellite cells and thus must be regulated to avoid premature satellite cell activation. MiR-31 is sequestered with Myf5 in mRNP granules where it ensures silencing and prevents Myf5 mRNA accumulation in resting satellite cells. Inhibition of miR-31 leads to Myf5 protein accumulation and cell cycle re-entry (75). Overall, quiescence is maintained in satellite cells through different mechanisms such as repression of cell cycle, regulation of intracellular pathways, and interactions with molecular and cellular components of the satellite cell niche. However, these quiescent satellite cells are primed or “poised” to be activated within a short period of time following muscle injury. Thus, quiescence is an active process and complex regulatory pathways are needed to guarantee efficient re-entry into the cell cycle upon muscle injury.

Satellite Cell Activation Quiescent satellite cells can quickly respond to changes in their niche or to specific signals sent by their microenvironment. For instance, the basal lamina contains molecules that can entrap growth factors. Following muscle injury, damage to the ECM releases these growth factors and some of them can interact with the satellite cells to stimulate their activation. FGF2 is one of these factors (88). FGF receptors are upregulated in activated satellite cells compared to quiescent satellite cells (153). The presence of Syndecan-4 on satellite cells is required to mediate FGF2 signaling (73). FGF2 induces a quick increase in intracellular calcium concentration through TRPC channels leading to NFATc translocation into the nucleus and satellite cell activation (181). FGF2 also

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activates the p38 MAPK pathway and inhibition of this pathway prevents satellite cell activation (144, 189). Other isoforms of FGF such as FGF6 have been shown to play a role in muscle regeneration through the stimulation of satellite cell activation and/or proliferation (99). Another critical growth factor for satellite cell activation is the HGF. Similar to FGF2, HGF is located in the ECM of uninjured muscle and is released following muscle injury (295). HGF binds to c-Met, a receptor expressed on quiescent satellite cells. Colocalization of HGF and c-Met was shown on satellite cells in regenerating muscle. In vitro HGF promotes satellite cell entry into the cell cycle (6). Similar to FGF, the presence of Syndecan-4 is necessary to facilitate HGF signaling (73). Local expression of IGF-1 using a muscle-restricted transgene showed that overexpression of IGF-1 can also activate satellite cells (210). In vivo, IGF-1 is produced by different local cell types such as fibroblasts and myofibers (231). IGF-1 is well known to stimulate the Akt/mTOR pathway and consequently downregulate the activity of the transcription factor FOXO. Downregulation of FOXO1 by IGF-1 was shown to inactivate the cell cycle repressor p27kip and induce cycling of satellite cells (187). Nitric oxide (NO) is a molecule almost instantly generated following damage. Inhibition of NO synthase, the enzyme responsible for NO production, was shown to decrease the immediate response of satellite cells to injury (8). It has been demonstrated that NO stimulates MMP expression and increases the release of growth factors from the ECM (294). A cytokine, TNF-α, is also quickly produced and released following muscle injury. Injection of TNF-α in healthy uninjured muscles results in satellite cell activation and entry in the cell cycle (176). Activation of the NF-kB pathway by TNF-α silences gene expression of Notch1, which results in satellite cell activation (3). Lipid mediator pathways are also involved in satellite cell activation, particularly Sphingosine-1-phosphate (S1P). S1P is released through the metabolism of sphingomyelin located on the inner surface of the plasma membrane. Sphingomyelin is abundant in the membrane of quiescent, but not proliferating, satellite cells (213). Consistently, S1P stimulation promotes satellite cell entry in the cell cycle while inhibition of this pathway decreases the response of satellite cells to mitogen stimulation (214). Altogether, these studies indicate that the exit from quiescence into a proliferative state is mediated by various extracellular signals released from the damaged muscle. A cocktail of cytokines and growth factors stimulates receptors present on the quiescent satellite cell membrane and activates different signaling pathways that promote cell cycle re-entry (Table 1).

Asymmetric Division of Satellite Cells Upon activation, Myf5pos committed satellite cells can proliferate and differentiate. In contrast, Myf5neg satellite stem

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Asymmetric self-renewal and differentiation

Stochastic self-renewal and differentiation

Satellite stem cell

Extracellular matrix

Asymmetric division

Muscle fiber

Symmetric division

Committed satellite cell

Figure 10 Asymmetric and stochastic self-renewal. Satellite cells can be divided in two subpopulations: committed satellite cells and satellite stem cells. Satellite stem cells can perform asymmetric division in an apical-basal orientation or symmetric division in a planar orientation (relative to the myofiber). Asymmetric division allows for the formation of one committed daughter cell that will participate in myogenesis, and for the maintenance of the original satellite stem cell. Symmetric division of satellite stem cells produces two daughter stem cells and favors satellite stem cell expansion. Committed satellite cells can divide to form two committed daughter cells that participate in the myogenesis process.

cells are able to self-renew through symmetric or asymmetric division upon entry into the cell cycle. Asymmetric divisions produce one daughter stem cell and one daughter committed cell (Fig. 10). Alternatively, symmetric divisions give rise to two identical daughter stem cells. Although the elements controlling satellite stem cell fate are not well understood, different factors and mechanisms of action begin to be unraveled. A long-standing question was how Myf5 expression is regulated in Myf5neg satellite stem cell during asymmetric division. Indeed, Pax7 is known to induce Myf5 expression and it is present in both Myf5pos and Myf5neg cells after

(A)

(B)

asymmetric division. Interestingly, it was demonstrated that Pax7 needs to be methylated by the arginine methyltransferase Carm1 to induce Myf5 transcription (155,195). During asymmetric division, in the Myf5neg satellite stem cells Pax7 does not interact with Carm1 and therefore Myf5 expression is prevented. The satellite stem cell niche has a critical influence on satellite stem cell fate. It has been shown that muscle stem cells dividing in an apical-basal position (perpendicular to the myofiber) perform mostly asymmetric divisions, while planar divisions (parallel to the myofiber) are symmetric (Fig. 11)

(C)

Figure 11

Symmetric versus asymmetric divisions. Images show myofibers isolated from EDL muscles of Myf5-cre R26R-YFP stained with Pax7 (red), YFP (for Myf5, green), and a nuclear dye (DAPI, blue). The myofiber membrane is outlined by a dashed line. In these mice, expression of Myf5 leads to permanent yellow-fluorescent protein (YFP) staining. Most of the satellite cells express Myf5 (YFP+) during development, but a subpopulation of satellite stem cells never expressed Myf5 (YFP−). YFP− satellite stem cells can perform asymmetric division (A) and give rise to a committed progenitor (YFP+), or perform symmetric division (B) and produce two satellite stem cells (YFP−). Asymmetric divisions occur mostly in an apical-basal orientation (A), while symmetric divisions occur with planar orientation (B and C).

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(163). Generally, during apical-basal asymmetric division, the cell that remains attached to the basal lamina stays Myf5neg while the cell that is pushed toward the myofiber becomes a Myf5pos committed progenitor. In vitro culture of myogenic progenitors in ECM-coated micropatterns showed that when a myogenic progenitor cell has symmetrical adhesion to the ECM it is most likely to perform random DNA segregation. On the other hand, a myogenic progenitor cell that has asymmetric adherence to the ECM will often perform nonrandom DNA segregation (326). On asymmetric ECM micropatterns the cell that retains the old template DNA is preferentially located in the low adhesion side while the committed cell localizes to the high adherence side. Interestingly, Pax7hi cells that intrinsically tend to perform asymmetric division are not affected by symmetric or asymmetric micropatterns, which suggests that intrinsic factors also have an influence on stem cell fate. However, it may be difficult to extrapolate these in vitro findings to in situ satellite cells. Nevertheless, these findings suggest that ECM adherence in the satellite cell niche could influence muscle stem cell fate decision. In asymmetric divisions, many different proteins have been shown to preferentially locate to either the mother or the daughter cell. The partitioning defective (PAR) complex comprising PAR-3, PAR-6, and atypical protein kinase C (aPKC) is polarized in the committed daughter cell and activates p38α/β MAPK asymmetrically (302). Activation of p38α/β MAPK leads to induction of MyoD and consequently induces commitment and proliferation of the daughter cell. The original template DNA that is asymmetrically located in the mother cell has been shown to colocalize with the Notch signaling inhibitor Numb (277). However, another study suggests that Numb is asymmetrically located in the committed daughter cell (70). Consistent with these latter results it has been shown that the mother stem cell expresses high levels of Notch-3 while the committed daughter cell highly expresses Notch ligand Delta-1 (163). Interactions between the mother and the daughter cell could then strengthen the cell fate of each cell. The stimulation of the Notch pathway in the mother cell is important to maintain its stemness as demonstrated by the increased commitment in the presence of Notch inhibitors (163). Overall, during asymmetric division, a variety of proteins are differentially distributed between the cells. Some proteins such as Notch-3 stay in the mother stem cell, while other proteins like PAR3-PAR6-aPCK complex and phosphop38MAPK are located in the committed daughter cell and trigger the myogenic program through Myf5 and/or MyoD activation.

Satellite Stem Cell Self-Renewal During muscle regeneration, the vast majority of satellite cells becomes activated, proliferates and differentiates to repair myofibers. However, to be prepared for subsequent injuries,

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skeletal muscle needs to replenish its satellite cell population. Importantly, even following multiple injuries, the satellite cell population remains constant (276). This capacity to restore the overall population is due to the high self-renewal ability of satellite cells. Transplantation experiments nicely illustrate this enormous self-renewing ability. Grafting of a single intact myofiber containing few satellite cells into a radiated muscle can regenerate up to a hundred myofibers (67). Similarly, a single transplanted satellite cell is able to undergo massive waves of proliferation leading to satellite cell repopulation (255). Importantly, the engraftment potential of the Myf5neg satellite stem cell subpopulation is much higher than the one of committed satellite cells (163). Symmetric self-renewal of Myf5neg satellite stem cells involves the polarized distribution of proteins during division. The planar cell polarity (PCP) pathway has been shown to regulate oriented divisions during development (118). The PCP pathway is a noncanonical Wnt pathway activated by the interaction of wingless (Wnt) ligands with Frizzled receptors (Fzd). Binding of Wnt to Fzd induces the cytoplasmic scaffolding protein Disheveled to activate Rac/JNK and Rho/ROCK pathways (158). These downstream pathways regulate cytoskeleton reorganization and gene expression. Analysis of mRNA expression from FACS sorted Myf5neg stem cells versus committed satellite cells revealed that Fzd7 is highly enriched in the stem cell subpopulation (169). Moreover, Wnt7a is upregulated during muscle regeneration and interacts with Fzd7 to mediate symmetric divisions of Myf5neg satellite stem cells (169) (Fig. 12). Fzd7 induces polarized expression of Vangl2, a well-known effector of the PCP pathway. Knockdown of Vangl2 reduces symmetric division and favors apical-basal asymmetric divisions. Planar symmetric expansion allows each daughter cell to receive equal feedback from their niche (e.g., from the basal lamina and the plasmalemma). Physical interaction with the microenvironment is critical to support self-renewal of satellite stem cells (163). For instance, the binding of Fzd7 to the ECM protein fibronectin through syndecan-4 coreceptor provides feedback and enhances Wnt7a/Fzd7-mediated symmetric proliferation of satellite stem cells (26). Collagen VI is another ECM component that is found in the satellite cell niche and that affects satellite cell self-renewal (304). Lack of collagen VI leads to impaired muscle regeneration and exhaustion of the satellite cell pool following multiple injuries. In addition to the symmetric expansion of satellite stem cells, it was also hypothesized that committed satellite cells can withdraw from terminal differentiation to replenish the satellite cell population. These observations were first shown in vitro using C2C12 myoblasts cultured in low-serum differentiation medium. While most of the myoblasts differentiate, a small subpopulation of myoblasts called “reserve cells” is unable to differentiate and shows greatly reduced MyoD and Myf5 expression (328). One must keep in mind that these cells are immortalized myoblasts that have different behavior than satellite cells in vivo. Nonetheless, culture of isolated

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Satellite stem cells FN

Symmetric cell division

Myogenic progenitors

Cell migration

Cytoskeleton

Muscle fibers

Muscle hypertrophy

Figure 12

Wnt7a-activated pathways in myogenic cells. In myogenic cells, Wnt7a binds to Fzd7 and induces various responses depending on the myogenic stage. In satellite stem cells, Wnt7a forms a complex with the Fzd7 receptor, Scd4 coreceptor and fibronectin to activate the planar cell polarity pathway. This noncanonical pathway leads to the symmetric stem cell division. In myogenic progenitors, Wnt7a leads to rearrangements of the cytoskeleton and increases directional cell migration. Lastly, in muscle fibers, Wnt7a activates the Akt/mTOR pathway, in an IGF-1-independent manner, and promotes myofiber hypertrophy.

myofibers ex vivo also showed that Pax7-positive activated satellite cells can lose MyoD expression and exit the cell cycle to maintain the satellite cell pool (329). These cells can potentially be reactivated in a future muscle injury. However, the mechanism of myoblast dedifferentiation remains to be clarified.

1038

Self-renewal of the satellite cell pool also depends on the ability of the cells to withdraw from cell cycle and return to quiescence. Indeed, it was observed that following muscle regeneration in the presence of the proliferation label BrdU, quiescent satellite cells were marked, which indicates that these cells had previously divided (266). Cell-cycle

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withdrawal is necessary for both differentiation or return to quiescence. Thus, the molecular mechanisms mediating cell fate decision need to be extraordinarily well defined to regulate both processes simultaneously. Microarray analysis of human satellite cells returning to quiescence versus proliferating satellite cells revealed substantial differences in gene expression (160). The authors of this study observed that there is an overall decrease in total mRNAs in quiescent satellite cells compared to proliferating cells. Consistently, the activity of RNAseL, an enzyme involved in RNA destruction, increases when satellite cells return to quiescence. MicroRNAs are also globally downregulated in satellite cells that become dormant (160). Regulation of the ERK1/2 pathway is important to mediate the return to quiescence. As discussed previously, fibroblasts and smooth muscle cells located in the microenvironment of activated satellite cells during muscle regeneration can secrete Ang-1 that binds to Tie-2 receptor on satellite cells. This interaction promotes cell cycle exit, represses differentiation and supports return to quiescence, through the ERK1/2 pathway (2). Moreover, the transcription factor Six1 has been shown to repress the ERK pathway by regulating Dusp6, a phosphatase that inhibits MAPK phosphorylation. Downregulation of Six1 or Dusp6 supports the return to quiescence and satellite cell niche occupancy (168). Moreover, the receptor tyrosine kinase inhibitor, Sprouty1, is upregulated when Pax7-positive cells re-enter quiescence to inhibit FGF2 signaling and induces cell cycle exit (274). Constitutively active Notch signaling has been shown to inhibit myoblast differentiation and promote satellite cell self-renewal (Table 1) (319). As previously discussed, Syndecan-3, a protein highly expressed in quiescent satellite cells, was shown to directly bind and activate Notch to promote satellite cell return to quiescence (233). Reactivation of Notch signaling is also driven by the FOXO3 transcription factor, and these two proteins are involved into a regulatory feedback loop (115). FOXO3 stimulates Notch1 and Notch3 receptor expression, while activation of Notch results in increased FOXO3 expression. FOXO is directly downregulated by the Akt/mTOR pathway. Therefore, during regeneration, the release of growth factors such as IGF-1 stimulates the Akt/mTOR pathway that inhibits FOXO and thus downregulates Notch. This cascade enables satellite cell proliferation and differentiation. The decrease in growth factors present in the microenvironment at the end of the regeneration process then appears to induce an increase in FOXO and Notch signaling that favors self-renewal and the return to quiescence. In summary, signaling pathways that promote the return to quiescence only begin to be unraveled and the significance of this process is likely underestimated (Table 1). Emerging evidence in the literature suggests that this important process is affected in aging and is gradually lost during various degenerative conditions (see section on pathologies and degenerative conditions).

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Myogenic Cell Migration Satellite cells are distributed all along the length of myofibers. Following muscle injury, activated satellite cells migrate from their original location to the site of the lesion (267). Real time video microscopy of satellite cells on isolated fibers ex vivo clearly illustrates the high migratory capacity of activated satellite cells (278). Graft experiments of labeled cells revealed that satellite cells can migrate from one fiber to another over many millimeters (142, 313). In vitro migration assays and in vivo transplantation experiments indicate that satellite cell migration capacity is higher before they start to proliferate (255, 278). Analysis of satellite cell activity following muscle injury demonstrated that activated satellite cells first migrate into the damaged area and then increase their mitotic activity at the site of injury (267). Adhesion molecules, soluble molecules, and guidance cues are important to direct migration of satellite cells (Fig. 13). Adhesion of myogenic cells to the ECM is important to mediate migration. Fibronectin and collagen are able to increase migration in vitro, but generally, laminin is considered the most potent inducer of satellite cell migration (278). Expression of integrin-α7 and -β1 on the satellite cell membrane allows them to bind to laminin. CD34, a membrane protein highly expressed in satellite cells also mediates cell motility (5). In CD34-deficient mice, the migration of satellite cells along the myofiber is decreased and muscle regeneration is impaired. Deficiency for CD44, another cell surface protein, leads to decreased motility of myoblasts and to a reduced response to soluble chemotactic molecules (212). CD44 can interact with various ECM proteins such as hyaluronan, collagens, fibronectin, or laminin (174). Although the ECM provides support for cell migration, it can also represent a physical barrier for migrating satellite cells especially in presence of fibrosis. The secretion of different Matrix Metalloproteinases (MMPs) by myogenic cells is important to facilitate their migration. MMPs are a family of enzymes that can specifically digest precise components of the ECM. For example, MMP-1 can specifically degrade collagen type I, II, and III to facilitate myoblast migration (147). MMP2 and MMP-9 are also upregulated during the early regeneration process and could degrade type-IV collagen that is enriched in the basal lamina (156). Overexpression of MMP9 was shown to increase migration of transplanted myoblasts (207). MMP activity is regulated by different enzymes, named Tissue Inhibitors of MMP (TIMPs). TIMP expression is usually increased during muscle regeneration and can positively or negatively affect MMP activity depending on the TIMP isoform and concentration (150). In vitro experiments demonstrated that MMP inhibition appears to decrease myoblast migration (217). Soluble molecules liberated after muscle injury are important to drive satellite cell migration toward the site of injury. Crushed muscle extract is able to mediate myoblast chemotaxis, by promoting directional migration along a gradient of attracting molecules (32). Among the different factors present

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Figure 13

Satellite cell migration. Schematic representing a migrating satellite cell. Satellite cells receive many guidance cues from their microenvironment that promote chemotaxis toward the damaged area (ex: HGF) or repulse the cell away from uninjured region (ex: Ephrins).

in the crushed muscle extract, TGF-β and HGF have been shown to directly increase the motility of myogenic cells. FGF-2 and FGF-6 can also stimulate satellite cell motility (215, 278). Wnt7a expressed during regeneration can guide myogenic cells by activating the PCP pathway through the Fzd7 receptor (Fig. 12) (23). Similarly, vascular endothelial growth factor (VEGF) affects chemotaxis of myogenic cells in regenerating muscle. Myoblasts express both VEGF and its receptors, a characteristic that could potentially allow for autoregulatory interactions (108). Interleukine-4 (IL-4) also stimulates myoblast migration by increasing the expression of specific integrins, such as β1-integrin (165). In addition to the various chemoattractant molecules that induce migration toward the site of injury, there are also molecules that have chemorepulsive effects. Activated satellite cells and muscle fiber both express Ephrin ligand and Eph receptor. Multiple Ephrins were shown to induce repulsion of satellite cells in vitro (286). Ephrins are expressed in healthy fibers and could thus favor migration of satellite cells away from undamaged fibers. Overall, satellite cells are highly motile during muscle regeneration. This migration capacity is mediated by a

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multitude of physical and chemical guidance cues. Migration of satellite cells is essential to direct myogenic precursors to the site of injury, where they can then proliferate and fuse to regenerate the damaged muscle.

Myoblast Proliferation Activated myogenic progenitors undergo several rounds of proliferation to increase the myogenic pool needed for tissue repair. Inhibition of cell proliferation makes muscle regeneration impossible (232). The majority of the growth factors known to activate satellite cells also facilitates cell division. Addition of FGF2 to the medium of primary myoblasts or in fiber cultures in vitro is commonly used to stimulate myogenic cell proliferation. However, FGF2 is not a mitogen and its effect on proliferation is probably due to increased satellite cell activation (323). Several studies have also revealed the role of IGF-1 for the stimulation of cell-cycle progression of satellite cells. Satellite cells from mice overexpressing IGF-1 have enhanced proliferative lifespan and can generate a much bigger myogenic cell population (54). In vitro

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experiments have shown that addition of low concentration of HGF promotes myoblast proliferation while higher concentrations lead to decreased proliferation (324). Following muscle damage, proinflammatory cytokines are enriched in the injured tissue. This boost in proinflammatory cytokines, such as IL-6, IL-1β, and TNF-α, coincides with the initiation of the proliferative phase of myoblasts (298). IL-6 can be produced by myoblasts or myofibers and acts in an autocrine or paracrine manner to stimulate myoblast proliferation (44,312). IL-6 deficiency reduces myoblast proliferation by impairing STAT3 signaling and its downstream target, the cell cycle protein cyclin-D1 (272). IL-1β and TNF-α during the early regenerative phase are also known to stimulate IL-6 expression (185, 236). Moreover, these factors can directly promote myoblast proliferation, possibly through the NF-kB pathway (220). Similar to NF-kB, the JNK pathway favors myoblast proliferation by promoting the transcription of cellcycle proteins like Cyclin-D1 (Table 1) (7, 124, 230). On the other hand, certain signaling pathways encourage proliferation by repressing premature differentiation. p38γ can directly phosphorylate MyoD leading to the formation of a repressive transcriptional complex that will occupy the myogenin promoter and repress premature activation (112). Similarly, JAK1/STAT1 promotes myoblast proliferation and inhibits premature differentiation by repressing differentiation genes such as MEF2 (289). Several microRNAs were shown to control the switch from proliferation to differentiation. For instance, miR-221 and miR-222 are more expressed in proliferating myoblasts than in differentiating myotubes (46). These two microRNAs repress the cell cycle inhibitor p27 and the differentiation factor myogenin, which results in delayed withdrawal from the cell cycle. MiR-133 also promotes myoblast proliferation by repressing the serum response factor (SRF), which is involved in myogenic differentiation and expression of myofiber-specific genes (56, 315). Overall, there is a multitude of different factors and signaling pathways involved in myoblast proliferation. These extrinsic factors promote myoblast proliferation either directly by increasing cell cycle progression and/or by repressing precocious differentiation. 0d

1d

Myogenic Differentiation Following several rounds of proliferation, myogenic cells exit the cell cycle and start to differentiate. Thereafter, myogenic cells fuse together to form multinuclear myotubes or fuse to existing damaged fibers. Different cues control this switch from the proliferation to the differentiation state. In vitro, myoblasts can be expanded in the presence of high concentrations of serum and switching to low-serum medium induces their differentiation and fusion to the neighboring cells (Fig. 14). MyoD is a key factor for myogenic progression and subsequent differentiation. MyoD−/− myoblasts fail to upregulate myogenin and MRF4, display differentiation defects and impaired muscle regeneration (72,198,254). MyoD facilitates the transition from myoblast proliferation to differentiation through the induction of cell cycle inhibitors such as p21 and p57, and by inducing myogenin expression (125, 132). Downstream of MyoD, myogenin triggers the expression of genes involved in muscle contractility such as myosin light chain, myosin heavy chain, muscle creatine kinase, α-actinin, troponin, and voltage-dependent calcium channel (80). Similarly, MRF4, an MRF with functions related to myogenin, is involved in late differentiation (242). MRF activity during differentiation is also controlled by the MEF2 family of transcription factors. For instance, MEF2C can physically interact with MyoD or myogenin and synergistically activate different muscle gene promoters (204). Moreover, there is a positive regulatory feedback loop between MEF2 and MRF expression. Therefore, forced expression of MyoD or myogenin can induce MEF2 expression, while overexpression of MEF2, in turn, increases MyoD expression and myoblast differentiation (76, 154). Conditional deletion of different MEF2 genes (MEF2A, MEF2C, and MEF2D) in satellite cells revealed that absence of only one isoform can be compensated by the others. However, combined deletion of the different MEF2 genes leads to impaired myogenic differentiation and muscle regeneration (180). Certain microRNAs are involved in the transition from myogenic cell proliferation to differentiation. For instance, miR-1, miR-133, miR-206, and miR-486, are upregulated 3d

5d

Figure 14

Myotube formation. Pictures from primary myoblasts cultured in vitro in differentiation medium (low-serum medium). Cells are stained with a nuclear dye (DAPI, blue) and myosin heavy chain (MyHC, in green). Almost no myoblasts express MyHC before the addition of differentiation medium. After 1 day in differentiation medium, the cells start to express MyHC. At three days, myoblasts fuse to form small multinucleated myotubes (few myonuclei per myotube). After 5 days of differentiation, myotubes become larger and contain more nuclei.

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during myoblast differentiation and myotube formation (159, 160). These microRNAs enhance differentiation by directly inhibiting Pax7 transcription, which consequently results in increased MyoD activity (57, 87). Distinct signaling pathways regulate MRF expression and thus affect the switch from myoblast proliferation to differentiation (Table 1). A switch from Notch to the canonical Wnt signaling pathway has been shown to be critical for the transition to myoblast differentiation (39). The cross-talk between these two pathways is mediated by the protein kinase Glycogen synthase kinase (GSK3). The MAPK signaling protein p38α is also involved in myoblast differentiation (321). p38α decreases myoblast proliferation by antagonizing the effect of the proliferation-promoting pathway JNK (230). Moreover, p38α stimulates MyoD transcriptional activity and consequently the transcription of muscle-specific genes (182). p38α also directly phosphorylates MEF2A and MEF2C, the latter being a coactivator of MyoD (321). Calcium-activated pathways have been shown to be involved in myogenic differentiation. Calcium is a major regulator of muscle cell function and is thus tightly controlled. Increased intracellular calcium concentrations lead to the rapid activation of calcineurin and calcium/calmodulindependent protein kinase (CaMK) pathways. Calcineurin activates MyoD through MEF2C (101). Inhibition of calcineurin leads to decreased myogenin expression and impaired differentiation (100). CaMK also increases MEF2 activity by removing the inhibitory action of HDAC proteins on MEF2 target gene expression (184). Thus, p38α and the calcium-activated pathways both promote in a nonredundant manner MyoD and MEF2 activities resulting in increased myogenin expression and myogenic differentiation (322).

Myogenic Cell Fusion and Myotube Formation Following the induction of the differentiation program, myogenic cells undergo a cell-cell fusion process that will strongly modify cell shape and function (Fig. 14). To fuse, myocytes must first recognize and adhere to each other. Myocyte fusion has mostly been studied in Drosophila because in this experimental system muscle formation is easily visualized. In Drosophila, interaction between Dumbfounded (Duf), Sticks and stones (Sns), Roughest (Rst), and Hibris (Hbs) proteins mediate cell recognition and adhesion (248). Duf and Rst both interact with Sns and have redundant function so that depletion of both proteins is necessary to cause a fusion deficit (288). Notch signaling downregulates the expression of adhesion proteins such as Sns, that are important for fusion (111). In mice, related adhesion molecules such as Nephrin (Sns homolog), β1-integrin, Focal adhesion kinase, M-cadherin, and N-cadherin have been described (1, 282). Upon myoblast-myoblast contact, Duf and Sns proteins form a ring-like structure called fusion-restricted

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myogenic-adhesive structure (FuRMAS) in Drosophila. The formation of this structure is hypothesized to be a key event leading to the recruitment of the molecular fusion machinery. Following FuRMAS establishment, actin filaments accumulate in the center of the fusion site. Rho GTPase family members such as Rac1 play an essential role to drive actin polymerization through activation of Actin related protein (Arp2/3) (287). During the next steps of the fusion process, intracellular perfusion vesicles accumulate at the contact site of the two adherent cells. Actin was hypothesized to partially mediate the recruitment of these vesicles but its exact role remains unclear (157, 248). Thereafter, one or many small pores in the membranes are created. These pores will then enlarge to eventually dissolve completely to give rise to a multinucleated cell. Importantly, re-expression of adhesion molecules by the newly formed multinucleated cell is necessary to allow further myocyte recognition and to continue the fusion process. Until recently, the proteins regulating myoblast fusion in mammals were elusive. However, a muscle-specific membrane protein named myomaker was shown to control myoblast fusion (200). Absence of myomaker in mice leads to perinatal death due to lack of multinucleated myofibers. Accordingly, inducible deletion of myomaker specifically in satellite cells (using pax7-creERT2 mice) strongly perturbs muscle regeneration (201). Interestingly, myomaker-deficient mice express normal level of MyoD and myogenin indicating that muscle precursor cells are able to commit into the myogenic lineage up to a certain point. Moreover, MyoD and myogenin were shown to induce myomaker transcription. Mechanistically, myomaker requires actin cytoskeletal rearrangement to induce fusion (200). The fusion process can be divided into early and late fusion. Early fusion involves myocyte-myocyte contact, while late fusion implies the fusion of myocytes to nascent multinucleated myotubes. Although they share many similarities, these two modes of fusion involve different adhesion molecules and signaling pathways. For example, β1-integrin mediates myocyte-myocyte fusion. Consequently, β1-integrin-deficient myoblasts are able to adhere to each other but cannot fuse (268). On the other hand, proteins such as the actin binding protein FilaminC are involved in myocyte-myotube fusion. In vitro, knockdown of FilaminC in differentiating myoblasts results in formation of small and round myotubes with limited numbers of nuclei (78). During late fusion, the calcium activated transcription factor NFATc2 is upregulated. NFATc2 controls the expression of IL-4. Myoblasts from IL-4-deficient mice are able to fuse but form smaller myotubes with less myonuclei (133). Thus, after initial myoblast-myoblast fusion, the nascent myotubes secrete IL-4 to recruit more myoblasts and perform late fusion. At this stage, interactions of myotubes with the ECM are particularly important to stimulate myotube formation. In vitro studies showed that laminin promotes myotube alignment and survival (63). Generally, the proteins and signaling pathways involved in myocyte fusion show some discrepancies between

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the different species but the major events during the fusion process are conserved.

Myotube Maturation and Myofiber Hypertrophy Nascent myotubes need to undergo a maturation process to become fully functional myofibers. Mature muscle fibers are highly specialized and need to acquire a competent excitationcontraction coupling, contractile, and metabolic machineries. The Akt/mTOR pathway has a pivotal role in myotube maturation and hypertrophy (Table 1) (225, 226). Interaction of IGF-1 with its receptor activates Akt and subsequently its downstream effector mTOR (249). In absence of endogenous mTOR, C2C12 myoblasts are able to fuse together, but they cannot form mature myotubes and express contractile proteins. The Akt-1/mTOR pathway induces various trophic effects via several pathways (18). mTOR activation stimulates protein synthesis by phosphorylating p70S6k that activates the ribosomal protein S6. mTOR also exerts anabolic effects by inhibiting the translation repressor 4E-protein binding 1 (4EBP1). Akt, in turn, has been shown to support hypertrophy by inhibiting the activity of FOXO, a factor involved in protein catabolism. FOXO can induce muscle ring finger-1 (MuRF1) and atrogin-1 expression, two E3-ubiquitin ligases strongly associated with muscle wasting (261). Although IGF-1 is the most well described factor activating the Akt/mTOR signaling, other proteins and growth factors were shown to induce this pathway. Interestingly, Wnt7a, which stimulates satellite stem cell expansion (see section on self-renewal), can also act directly on myotubes and induce a hypertrophic response through the activation of the Akt/mTOR pathway (Fig. 12) (308, 311). Surprisingly, long-term overstimulation of mTOR induces a strong feedback mechanism characterized by a powerful induction of E3-ubiquitin ligases and muscle atrophy (21). Thus, Akt/mTOR pathway needs to be tightly regulated to stimulate muscle hypertrophy. It has been suggested that increases in calcium signaling can stimulate muscle fiber hypertrophy. However, these results are still controversial (141, 227, 249). The main function of calcium-dependent signaling pathways, such as the Calcineurin and CaMK cascades, in differentiated fibers appears to be the determination of muscle fiber type (e.g., slow vs. fast). Briefly, Calcineurin activation leads to NFAT translocation in the nucleus, which stimulates slow fiber gene transcription. Similarly, CaMK liberates and activates MEF2 transcription factors that promote the slow fiber gene program. Muscle fiber phenotype and plasticity have been extensively reviewed by Blaauw B and colleagues (34).

Epigenetics The previous sections illustrate that myogenesis is a complex and coordinated process relying on various extracellular cues and intracellular pathways. In addition to these diverse signals

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there is also other intrinsic factors that will determine satellite cell fate during myogenesis. During the last few decades, epigenetics emerged as a new field of research and many different studies indicated its crucial effect on myogenesis. Epigenetics involve modifications in gene expression that occur in absence of any modification of the primary DNA sequence. These alterations in transcriptional potential can be mediated by a multitude of external cues. Epigenetics include dynamic and reversible changes in the chromatin structure due to histone modifications or nucleosome positioning, as well as DNA methylation. Epigenetic changes can turn on or turn off genes and thus influence which protein will be expressed. Histone modifications can be associated with active gene expression, such as histone acetylation (H3Ac and H4Ac) or trimethylation of histone H3 lysine 4 (H3K4me3). Alternatively, other modifications are associated to gene repression, including trimethylation of histone H3 lysine 27 (H3K27me3) and methylation of lysine 9 of histone H3 (H3K9me) (Table 2) (335). Combinations of these various modifications have different consequences on gene activity, particularly modifications taking place on histone tails, known as the “histone code.” The study of stem cell epigenetics is technically challenging, because adult stem cells are very limited in numbers and do not represent a uniform population in terms of cell-cycle Table 2 Histone Marks Present on Different Groups of Genes During Myogenic Progression

Cell type

Family of genes (example)

Histone mark

Embryonic stem cells

Myogenic developmental regulators (Pax7)

H3K27me3 (repressive), H3K4me3 (active)

Quiescent satellite cells

Nonmyogenic developmental regulators (Neurog1) Satellite cell-specific (Pax7) Muscle-specific (Myog)

H3K27me3 (repressive), H3K4me3 (active)

PAX7 target genes (satellite cell-specific) (Myf5) Muscle-specific (Myog)

H3K4me3 (active)

Satellite cell-specific (Pax7) Muscle-specific (Myog)

H3K27me3 (repressive)

Proliferating myoblasts

Myotubes

H3K4me3 (active) unmarked (not H3K4me3, not H3K27me3)

H3deAc, H4deAc, H3K9me2, H3K27me3 (all repressive)

H3Ac, H4Ac, H3K4me3 (all active)

In embryonic stem cells and in quiescent satellite cells, genes encoding developmental regulators possess bivalent (repressive and active) histone marks. In quiescent satellite cells, stemness genes like Pax7 are marked by active chromatin modifications, whereas muscle-specific genes are mostly unmarked. In proliferating myoblasts, satellite cellspecific genes like Myf5 are active, while muscle-specific genes possess repressive histone marks. When cells differentiate into myotubes, satellite cell-specific genes are repressed, whereas muscle-specific genes like myogenin (Myog) present active chromatin marks.

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status and in their ability to regenerate a given tissue. Therefore, technical issues limit the usage of classical epigenetic approaches like chromatin immunoprecipitation (ChIP) and ChIP-seq (sequencing) for studying satellite cell epigenetics. As a consequence, most studies on satellite cell epigenetics have been performed in myoblasts. Thus, because myoblasts are proliferating whereas satellite cells are mostly quiescent, these experiments do not perfectly reflect the biology of satellite cells, particularly for the regulation of quiescence and selfrenewal. Recently, ChIP-seq and RNA-seq techniques were adapted for their use with small numbers of cells (4, 292). These techniques will open new perspectives in the study of stem cell epigenetic regulation.

Epigenetics of Satellite Cells and Myoblasts: Repression of a Muscle Genetic Program Satellite stem cells present a particular epigenetic state known as “poised” state. Many genes encoding developmental regulators possess at the same time some repressive (H3K27me3) and some active (H3K4me3) histone marks. This coexpression of active and repressive marks is observed in embryonic stem (ES) cells for the Pax3, Pax7, Myod1, and Mrf4 loci (171, 334). The coexistence of negative and positive (“bivalent”) histone modifications is thought to keep developmental regulators in a silenced state, keeping them poised for rapid activation upon reception of appropriate cues to differentiate along a specific lineage. In quiescent satellite cells, many developmental regulators important for nonmuscle lineages, including a large number of transcription factors, possess bivalent chromatin marks, consistent with the stemness characteristics of satellite cells (179). In fact, quiescent satellite cell chromatin is mostly in a permissive conformation, with about half of the annotated genes being marked with the active H3K4me3 mark. Satellite cell activation is characterized by the retention of the H3K4me3 mark and the acquisition of the repressive H3K27me3 mark on specific loci (Table 2) (179). In agreement with the previous findings, ChIP-seq analysis in myoblasts demonstrated that many genes that are almost not expressed in myoblasts, but are upregulated in myotubes, display RNA polymerase II (PolII) binding. The presence of an enzyme involved in RNA transcription on inactive genes reflects that these genes are in a poised state prior to their maximal expression (13). To prevent premature differentiation, satellite cells and myoblasts must possess a regulatory system that represses muscle gene expression. DNA methylation is one important mechanism that represses differentiation. Indeed, treatment with 5-azacytidine, a DNA methyltransferase inhibitor, has been shown to induce fibroblast transdifferentiation into myoblasts (296). In undifferentiated myoblasts, genes that are not expressed mainly possess repressive histone marks in the vicinity of their transcriptional start site (TSS). Core histones H3 and H4 are hypoacetylated and methylated

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at specific lysine residues (H3K9me2, H3K27me3) (13, 47, 106, 190, 330). Repressive histone marks are also found in quiescent satellite cells from old mice, whereas in young mice the frequency of H3K27me3 mark is relatively low (179). These repressive histone marks are deposited by histone deacetylases (HDACs) and histone methyltransferases (HMTs). HDACs are often found in large protein complexes together with HMTs and are recruited at chromatin regulatory regions of inactive muscle genes (47, 305, 330). In proliferating myoblasts, class II HDACs are found, together with the HMT SUV39H1, at the myogenin gene promoter (Fig. 15). Their presence is associated with hypoacetylation of histone H3 and methylation of H3K9 (190, 330). Reducing Suv39h1 levels using siRNA leads to muscle gene expression (190). HDACs also suppress myogenic differentiation by being recruited to muscle gene promoters via interaction with MEF2 and MyoD (184, 190, 240). Moreover, the histone deacetylase SIRT1 is expressed in proliferating myoblasts and its expression decreases as the cells differentiate into myotubes (Fig. 15) (106). SIRT1 is present on muscle regulatory elements and its downregulation using shRNAs results in increased muscle gene expression and myogenic differentiation (106). HDACs and HMTs also have the property to modify nonhistone proteins, including transcription factors. For example, MyoD is acetylated by PCAF, and deacetylated by HDAC1 and SIRT1 (106,190,263). Similarly, MEF2 is acetylated by p300 and deacetylated by SIRT1 (186, 333). One of the most studied methyltransferase complexes is the Polycomb Repressive Complex 2 (PRC2). Its catalytic subunit, the HMT EZH2, is highly expressed during development and in proliferating satellite cells, and is downregulated when myogenic differentiation is induced (47). In undifferentiated myoblasts, EZH2 is found at the regulatory regions of muscle specific genes together with HDAC1 and the transcription factor YY1 (Fig. 15). These genes are marked by H3K27me3 (13, 47). Conditional deletion of Ezh2 in satellite cells compromises satellite cell proliferation and differentiation, with an impact on muscle regeneration (145). Mice with a conditional deletion of Ezh2 have reduced muscle mass and present smaller myofibers. In addition, deletion of Ezh2 in satellite cells leads to derepression of genes that are normally expressed in nonmuscle lineages, underscoring the importance of EZH2 in establishing satellite cell transcriptional program (145). An alternative epigenetic modification is to replace canonical histones with specific histone isoforms that are known to induce repressed or activated chromatin. For example, histone H1b and its interacting homeodomain partner MSX1 are present in a Myod1 regulatory enhancer, where they induce a repressive chromatin state and inhibit muscle differentiation (170). In contrast, the presence of histone H3.3 in the Myod1 promoter is associated with MyoD expression and epigenetic memory (216). The role of specific histone isoforms in satellite cell epigenetics is still unexplored and it would be of great interest to expand our knowledge in this field.

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Lineage determination genes PRC2

Suv39H1 HDAC

Undifferentiated satellite cells

Stemness genes

SIRT1

SWI/SNF

MLL

HAT

HDAC

RNA pol II

YY1

YY1 HDAC MEF2

MyoD

For example, Pax7

For example, Myogenin Relocalization of chromatin modifiers

UTX MLL

SWI/SNF HAT

PRC2 RNA pol ll

Differentiating satellite cells MEF2

MyoD

For example, Myogenin

HDAC

For example, Pax7

Figure 15 Epigenetic control of stemness and lineage determination genes during myogenic differentiation. In undifferentiated satellite cells and myoblasts, lineage determination gene regulatory regions such as the myogenin promoter are repressed through a combination of epigenetic mechanisms. MyoD is repressed by its association with SIRT1 and HDACs. MEF2 also recruits HDACs as well as SUV39H1, the histone methyltransferase responsible for H3K9 methylation. The Polycomb Repressive complex 2 (PRC2) containing the histone methyltransferase EZH2 is also recruited on the myogenin promoter by the transcription factor YY1. Together, these protein complexes are responsible to establish heterochromatin formation that is repressive for transcription. In undifferentiated satellite cells and myoblasts, stemness genes like Pax7 are active and are thus occupied by histone acetyltransferases (HAT) as well as the SWI/SNF chromatin remodeling complex and the MLL/Trithorax complex, responsible for H3K4me3 deposition. These proteins promote the formation of euchromatin, which is permissive for transcription. When cells receive differentiation cues, these epigenetic complexes are relocalized to different regulatory regions. Repressive complexes leave the promoters of lineage determination genes, which become active, and could be recruited to the promoters of stemness genes that need to be silenced. In contrast, proteins involved in active chromatin configuration leave the promoters of stemness genes and are relocalized at muscle specific promoters (myogenin). The myogenin promoter is thus occupied by the MLL/Trithorax complex, by HATs, by the SWI/SNF complex as well as by the demethylase UTX.

While muscle specific genes need to be repressed in satellite cells, other genes important for satellite cell function need to be expressed. Pax7 is an important transcription factor that controls genetic programs taking place in satellite cells. Recent ChIP-seq and gene expression data analysis determined that Pax7 activates genes involved in myoblast growth and proliferation whereas it represses genes involved in myogenic differentiation (283). Pax7 cooperates with the Trithorax methyltransferase complex through direct interaction with the MLL2 subunit (155). Trithorax complex activates gene expression by methylating the lysine 4 of histone H3. In myoblasts, Pax7 is responsible for the recruitment of the Trithorax complex to the Myf5 promoter, a known Pax7 target gene, to mark the gene with H3K4me3 (195). It is likely that PAX7 activates the expression of its other target genes involved in the promotion of cell proliferation by a similar mechanism (283).

Muscle Differentiation: Reconfiguration of the Chromatin When satellite cells receive prodifferentiation cues, the chromatin structure close to muscle-specific genes needs to be remodeled to be permissive for transcription (Fig. 15). The repressive marks on muscle regulatory regions need to

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be removed and replaced by active histone modifications (Table 2). For example, H3K27 becomes hypomethylated at specific muscle regulatory regions after induction of myogenesis (47). EZH2, the protein responsible for methylation of H3K27, is downregulated at the transcriptional level as well as by the action of specific microRNAs (47, 146, 320). The demethylase UTX mediates the removal of the repressive H3K27me3 marks upon activation of the myogenic program (270). Following induction of myogenic differentiation, the HMT SUV39H1 dissociates from MyoD at muscle gene promoters that show reduced methylation at histone H3 lysine 9 (H3K9) (190), which is indicative of gene activation. The histone demethylase JMJD2A is recruited to the myogenin promoter following induction of differentiation, where it catalyzes the removal of the H3K9 methylation mark (306). Accordingly, following promyogenic signals, HDACs leave the promoters of muscle-specific genes and the histone tails surrounding these genes become acetylated (330). The expression of the deacetylase SIRT1 is also decreased as cells differentiate, consistent with an increased acetylation at muscle regulatory regions (106). Another elegant mechanism for the downregulation of HDAC activity at myogenic promoters is the titration of HDACs to other protein complexes. For example, HDAC1 interacts with MyoD and is recruited on muscle gene promoters in proliferating myoblasts. Upon induction of myogenesis, the transcription factor pRb is

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dephosphorylated, which promotes the formation of a complex between pRb and HDAC1 that coincides with the disassembly of the HDAC1-MyoD complex (240). It is interesting to note that pharmacological inhibition of HDACs can enhance muscle differentiation (138). Histone acetyltransferases (HATs) are recruited at specific muscle gene loci by different transcription factors to promote histone acetylation of their regulatory elements (197). Nucleosomes need to be distributed in a specific manner to provide access of DNA regulatory elements to the transcription factors and machinery. Nucleosome positioning is regulated by chromatin remodeling complexes that mobilize nucleosomes following ATP hydrolysis, but can also be determined by transcription factors binding to specific loci. Some “pioneer” transcription factors can penetrate repressive chromatin to bind their regulatory elements and initiate chromatin opening (62). One example is PBX1/MEIS which is constitutively bound to muscle regulatory regions even in the absence of differentiation cues (27, 82). PBX1/MEIS interacts with MyoD and could recruit MyoD to specific genes from the muscle lineage, even in the context of repressive chromatin, thus establishing myogenic potential (27). This may be a mechanism through which MyoD has the potential to mediate “myogenic conversion,” for example, by reprogramming the genome of a nonmuscle cell toward the expression of musclespecific genes (317). Transcription factor binding to a specific DNA element facilitates access to secondary transcription factors on adjacent sites. Alternatively, transcription factors can recruit chromatin remodeling enzymes that displace nucleosomes and facilitate transcription factor recruitment. MyoD interacts with the SWI/SNF chromatin remodeling complex and could recruit this complex on muscle regulatory elements (Fig. 15) (81, 82). The arginine methyltransferase PRMT5, which dimethylates histone H3 arginine 8 (H3R8me2), as well as the Scaffold Attachment Factor B1 (SAFB1), are also involved in the binding of the SWI/SNF complex at the myogenin promoter (77, 130). Recruitment of the SWI/SNF complex, in turn, is required for the binding of other myogenic transcription factors including MyoD itself, myogenin and MEF2 (82).

Signaling to chromatin during muscle differentiation The p38 MAPK is an important mediator of muscle cell differentiation. Signaling by p38 regulates muscle gene expression by different epigenetic mechanisms. p38 is detected by chromatin immunoprecipitation on the regulatory elements of muscle genes (279), where it phosphorylates multiple targets. One direct target of p38-mediated phosphorylation is the ubiquitous bHLH protein E47. Phosphorylation of E47 promotes its heterodimerization with MyoD and increases the transcriptional activity of E47/MyoD that is essential for myogenesis (182). MEF2 proteins are also directly phosphorylated by p38, with an increased transcriptional activity as a result (126, 332). In addition, phosphorylation of MEF2D by

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p38 mediates the recruitment of Trithorax methyltransferase complexes to MEF2D target genes, which methylates histone H3 lysine 4 (H3K4me3), a chromatin mark associated with active gene expression (241). The SWI/SNF chromatin remodeling complex is also directly targeted by p38, since the BAF60 subunit of the complex is phosphorylated by p38, promoting the recruitment of the SWI/SNF complex on myogenic regulatory regions and favoring gene expression (279). On the other hand, genes important for satellite cell proliferation and expansion need to be repressed upon p38-induced muscle differentiation. One example is the Pax7 gene that is silenced by the action of the PRC2 complex. EZH2, the enzymatic subunit of the complex, is phosphorylated by p38, which promotes the formation of repressive chromatin on the Pax7 promoter (222). It appears that during myogenic differentiation, EZH2 (and possibly other proteins involved in chromatin repression) leaves the promoters of muscle-specific genes to associate with and repress genes important for satellite cell proliferation and expansion. How these changes are regulated by p38 and other signaling pathways remains to be determined. Blockade of p38 can efficiently repress muscle gene transcription, however the recruitment of muscle-regulatory transcription factors or acetyltransferases to muscle specific loci is not compromised (279). These observations suggest that other signaling pathways converge to chromatin to activate muscle gene expression. One example is the IGF1/PI3K/AKT pathway that promotes the association between MyoD and the acetyltransferases p300 and PCAF (271). p300 is a direct target of phosphorylation by AKT1/2 (271). The calciumdependent pathway CaMK is another important player in muscle differentiation. CaMK signaling disrupts the interaction between HDAC4/5 and MEF2, allowing MEF2 to recruit coactivators and chromatin modifiers to activate gene transcription at MEF2 target genes (184, 330). In addition, HDAC4/5 are directly phosphorylated by CaMK, and this phosphorylation drives the nuclear export of HDAC4/5 to the cytoplasm, preventing the binding of HDAC to myogenic gene promoters (196). In addition to the different pathways described here, there are many more signaling events that must converge to the nucleus where the ultimate effect is the remodeling of chromatin to modify gene expression. The specific effect of these pathways on epigenetic regulation remains to be further investigated.

Inflammatory processes Satellite cells are essential for muscle regeneration, however efficient muscle recovery relies on many other cell types (Fig. 16). Muscle damage initially triggers a complex inflammatory process. This inflammatory reaction involves the interaction of resident and infiltrating inflammatory cell types, for example, leukocytes. Inflammation following muscle injury has long been known for its role in the clearance of cell debris and/or pathogens. However, leukocytes have also been shown

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Committed satellite cells

Blood vessel Vessel-associated cell types

Basal lamina Muscle fiber

Muscle fiber nucleus

Specialized ECM

Growth factors

Directly interacting cell types

Satellite cell

Cell types secreting ECM and growth factors

Fascia

Figure 16 The satellite cell microenvironment. The satellite cell niche is composed of various ECM proteins and cell types. Cell types such as inflammatory cells and stromal cells can physically interact with satellite cells or release various cytokines, growth factors and ECM components that will influence satellite cell behavior.

to play significant roles in regulating satellite cell behavior (Fig. 17). The inflammatory process following muscle damage can be divided in three major steps: initiation, development, and dampening. In the earliest stage after injury the different leukocytes residing in the tissue, for example, mast cells, macrophages and a subset of “patrolling” circulating monocytes, sense the perturbation of tissue homeostasis (14, 107). Damaged myofibers release molecules called damaged-associated molecular pattern (DAMPs) such as DNA, RNA, metabolites, and others that will be recognized by resident leukocytes. Mast cells are the first cell type to be activated in the course of this process. These cells can release preformed and newly synthesized cytokines such as TNF-α, tryptase, and IL-6 within minutes. At physiological concentration, these cytokines are known to stimulate satellite cell activation and proliferation (58, 91, 272). In vitro coculture experiments confirmed that

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factors secreted by mast cells enhance myoblast proliferation (90). In vivo, the inhibition of mast cell secretion delays muscle regeneration (93). Thus, the initiation of the inflammatory process is closely linked to the activation of satellite cells. Next to the activation of satellite cells, the burst of cytokines released by the resident leukocytes also leads to the recruitment of blood-circulating leukocytes. Granulocytes, such as neutrophils and eosinophils, are the first nonresident cell types to invade the injured muscle. Granulocytes possess a high phagocytosis potential and have a critical role in clearing muscle debris. The proinflammatory environment that granulocytes generate influences myogenesis. Indeed, an in vitro study suggested that oxidative stress induced by neutrophils can exacerbate membrane damage on myotubes (234). However, it appears that the effect of neutrophils on myogenesis is generally dependent on the injury type (92,235). At the opposite, eosinophils have recently been demonstrated to promote

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Myofiber growth Differentiation/fusion Proliferation Activation

Inflammatory cell activation

Myogenic stages


MC

Injury

PMN

1d

M1

3d

M2

5-7d

Figure 17

Inflammation and myogenesis. Inflammation is characterized by the activation of mast cells (MC), followed by the early recruitment of granulocytes (polymorphonuclear cells, PMN), and the accumulation of monocytes/macrophages. Macrophage accumulation is classically composed of an initial proinflammatory phase (M1 macrophages) followed by an anti-inflammatory phase (M2 macrophages). These different leukocytes secrete factors that directly influence the function of myogenic cells. Initial burst of leukocytes has been shown to stimulate satellite cell activation and proliferation. M1 macrophages promote myoblast proliferation and repress early differentiation, while M2 macrophages stimulate differentiation and myofiber growth.

granulocytes and favor nonphlogistic phagocytosis (e.g., that does not stimulate inflammation) of apoptotic neutrophils (281). In addition to dampening of the inflammatory reaction, cytokines and growth factors such as IL-4, IL-10, and IGF-1 released by M2 macrophages also influence myogenesis (11, 183). IL-4 promotes myotube formation during the late fusion of myoblasts into myotubes (133). IGF-1 stimulates myotube hypertrophy (94). Consistently, the suppression of M2 macrophages in vivo leads to reduced myogenin expression and impaired fiber growth (86, 253). In regenerating human muscle, it has been shown that M2 macrophages are located in close proximity to myocytes expressing myogenin (257). Physical contact appears to increase macrophage-induced myogenic effects through a specific set of adhesion molecules (284). This proximity might also be important to allow macrophages to reconstitute the satellite cell niche through the secretion of different ECM proteins. M2 macrophages were shown to secrete a high amount of fibronectin and collagen VI, two proteins involved in satellite cell self-renewal (26, 117, 265, 304). Taken together, dampening of the inflammatory reaction in later stages after injury promotes myogenesis by facilitating differentiation, fusion and self-renewal.

Nonsatellite cell types with myogenic potential muscle regeneration (129). IL-4 secreted by eosinophils indirectly promotes myogenesis by stimulating fibro-adipogenic progenitors. The proinflammatory environment that neutrophils generate also encourages the recruitment of monocytes. Two subsets of monocytes accumulate in muscles following injury, the classical monocytes (Ly6C+) and the nonclassical monocytes (Ly6C-) (11). Classical monocytes are predominant during the first few days after an injury and are known to have proinflammatory phenotype. Classical monocytes secrete higher level of inflammatory factors such as TNF-α and IL-1β. This cocktail of proinflammatory molecules promotes myoblast proliferation and delays differentiation (11, 298). Thereafter, nonclassical monocytes become the main monocyte population present in the damaged tissue. These cells rather release anti-inflammatory molecules such as IL-10 and TGF-β (11). Nonclassical monocytes decrease myoblast proliferation and stimulate differentiation and fusion (298). Thus, the inflammatory reaction directed by leukocytes regulates satellite cell activation, proliferation, and differentiation (Fig. 17). In the course of the inflammatory process, infiltrated monocytes will differentiate and mature into macrophages. The local microenvironment polarizes macrophages into two different subpopulations, proinflammatory M1 macrophages and anti-inflammatory M2 macrophages (116). M2 macrophages are critical for mediating the resolution of inflammation. Anti-inflammatory molecules and proresolving mediators released by these cells will stop the further recruitment of

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Distinct ECM proteins influence the function of satellite cells during muscle regeneration. Connective tissue fibroblasts residing in the interstitium between myofibers are major producers and organizers of ECM in skeletal muscle (Fig. 16). Fibroblasts strongly proliferate following muscle injury and their expansion closely correlates with the increase in satellite cell numbers (209). Partial depletion of fibroblasts during muscle regeneration leads to decreased numbers of satellite cells and premature differentiation of myoblasts. Another cell type that participates in ECM formation is the fibroadipogenic progenitors (FAPs) that can give rise to fibrocytes or adipocytes. FAPs were shown to proliferate during muscle injury, but contrary to fibroblasts they promote myogenic differentiation instead of proliferation (143). This myogenic effect of FAPs could be mediated by the release of different myogenic factors, such as IGF-1, or by increased phagocytic debris removal (129, 143). Certain cell types that participate in myogenesis can also acquire Pax7 expression and become satellite cells (224). Heterogeneous population of cells with hematopoietic potential resident in the bone marrow as well as in muscle tissue called side population (SP) cells can give rise to satellite cells and contribute to muscle fibers upon transplantation (12,123,211,291). Lineage tracing of SP cells has shown that they display a strong proliferative response following muscle injury but only marginally contribute to the formation of myofibers (89). Germline Abgc2 knockout mice do not have SP cells in muscle and display impaired adult myogenesis, reduced satellite cell numbers, and fewer immune cells during

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regeneration (89). This suggests that under physiological conditions SP cells could have an accessory function for adult myogenesis by regulating the immune response. Mature muscle tissue also contains a population of Pax7 negative and PW1 (PEG3) positive interstitial cells termed PICs (PW1+ interstitial cells) (203). Upon transplantation, PICs engraft into muscle, contribute to muscle fibers and can generate Pax7-positive satellite cells. The population of PICs with myogenic potential in adult muscle has been shown to be Sca1 and CD34-positive but Pax7-negative. Importantly, freshly sorted PICs can acquire Pax7 and MyoD in vitro when cultured with myoblasts (203). Acquisition of a myogenic fate by PICs appears to be dependent on the induction of Pax7 expression. These results suggest that PICs represent a bipotent resident stem cell in skeletal muscle. Similar to other tissues, the microvasculature in adult skeletal muscle contains a population of contractile cells called pericytes that are wrapped around endothelial cells (114). Pericytes isolated from muscle do not express myogenic markers or endothelial markers but are positive for neuro-glial 2 proteoglycan (NG2), platelet derived growth factor receptor-beta (PDGFR-β), alpha-smooth muscle actin (αSMA) and alkaline phosphatase (AP) (85). In vitro, pericytes can differentiate into muscle fibers and coculture with myoblasts enhances this process. Pericytes can be expanded with high efficiency ex vivo and contribute to muscle fibers and satellite cells after infusion. Lineage tracing showed that pericytes, but not endothelial cells, are able to fuse to developing myofibers and contribute to myogenesis (85). This study also revealed that pericytes expand after muscle injury and that a relatively low number of pericytes can engraft in the satellite cell compartment. In addition to the above cell types that are present in the stem cell micro- or macroniche, other local or exogenous cell types such as CD133 and Integrin-β4 positive cells have been demonstrated to have myogenic potential (177, 260, 300). However, a role of these cell types during muscle regeneration remains to be determined. Moreover, as mentioned earlier, several ablation studies indicated that satellite cell depletion completely impairs muscle regeneration (see section on satellite cell and regeneration) (172,209,259,309). Consequently, satellite cells remain the principal myogenic precursor cells and no other cell type can compensate for their loss. On the other hand, in vitro experiments showed that nonsatellite cell types support adult myogenesis through indirect mechanisms that require coculture with myoblasts to participate to myotube formation. Thus, the absence of satellite cells in ablation experiments could also have indirectly affected the myogenic potential of nonsatellite cells. Overall, many cell types can contribute to myogenesis either indirectly by interacting with satellite cells or potentially by directly fusing to myofibers or engrafting into the satellite cell niche. Such cell types possess an important therapeutic potential and are currently under investigation by many groups.

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Pathologies and Degenerative Conditions Aging The regenerative capacity of skeletal muscle decreases slowly with aging. Regeneration in aged injured muscle results in more fibrotic tissue, increased intramuscular fat accumulation, and smaller myofibers (119, 258). Aging is also accompanied by a decrease in satellite cell numbers (275). This effect varies depending on the species, the age, the type of muscle, and the markers used (40). In addition to the exhaustion of the satellite cell pool, the myogenic capacity of individual satellite cells also becomes impaired during aging. For instance, in vitro culture revealed that aged myoblasts are able to proliferate and differentiate albeit to a slower rate than younger myoblasts (16,55). Whether this decrease in satellite cell function is fundamentally intrinsic, or extrinsically induced by the changing environment, remains a matter of debate. Increasing evidence suggests that both processes are involved (40). Extrinsic influences affecting satellite cells during aging can be systemic (ex: changes in hormonal levels), or local (ex: muscle fibrosis). In vitro primary myoblasts exposed to the serum of young mice have a higher myogenic potential than cells treated with old serum (49). Moreover, it has been shown by transplantation experiments that the age of the recipient defines the regenerative capacity of satellite cells (48). Therefore, old muscles that were transplanted into a young recipient have a higher regenerative capacity than young muscles transplanted into an old recipient. Importantly, it has been shown that aged muscles fail to increase levels of the Notch ligand Delta during muscle injury (68). As previously discussed, Notch has multiple essential roles in satellite cell activation, proliferation, and self-renewal (Table 1). Parabiotic pairings (e.g., a shared circulatory system between two mice) has revealed that Notch signaling can be restored in aged satellite cells by exposure to young serum (69). These results demonstrate that under certain conditions the satellite cell environment can partially overcome possible intrinsic defects in aged satellite cells. Old muscles were also shown to secrete excessive amounts of TGF-β, which overactivates pSmad3 and impairs satellite cell function (50). Interestingly, the pSmad3 and Notch pathways appear to antagonize each other. Activated aged satellite cells also exhibit a higher canonical Wnt signaling activity, which is potentially mediated by increased Wnt protein levels in the serum (40). Canonical Wnt signaling in old activated satellite cells results in the conversion of myogenic cells to fibrogenic cells. These results clearly illustrate the importance of the micro- and macroenvironment in the regulation of satellite cell myogenic potential. It has been suggested that a loss of satellite cell quiescence is responsible for the exhaustion of satellite cell pool and the reduced regenerative potential of aged muscles. Aged muscle fibers were shown to express more FGF2, which activates satellite cells and decreases their self-renewal potential (53). Moreover, loss of self-renewal in old satellite cells has

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been demonstrated to arise from an impaired FGF receptor response and increased p38α/β signaling (28). Increased p38α/β signaling in aged satellite cells has also been shown to stimulate the expression of senescence markers (74). Loss of reversible quiescence in aged satellite cells is accompanied by derepression of the CKI Cdkn2a (285). The above studies provided evidence that loss of quiescence and self-renewal is irreversible and cannot be restored completely by a youthful environment. Recently, it has been shown that the JAK/STAT signaling pathway is upregulated in aged satellite cells (237, 299). Increased Stat3 signaling is at least partially mediated by elevated level of IL-6 inflammatory cytokine (299). Stat3 overexpression leads to increased asymmetric division of satellite stem cells and promotes myogenic lineage progression through MyoD1 expression. Increased asymmetric division is correlated with an exhaustion of satellite stem cell subpopulation (237). Knockdown of Jak2 and/or Stat3 improves satellite stem cell symmetric division and increases the selfrenewal potential upon transplantation. Moreover, pharmacological inhibitors of Jak/Stat improve muscle regeneration and muscle force. Telomere shortening associated with aging has been hypothesized to be responsible for cellular senescence, e.g. the cessation of cell division caused by aging (43). A correlation between telomere length and satellite cell proliferative potential has been described (83). However, human satellite cells show only marginal signs of telomere shortening during aging (84). Thus, the exact role of telomere shortening on aged satellite cell behavior remains to be determined. Taken together, satellite cell functions rely on a fine-tuned balance of both intrinsic and extrinsic factors that can be disturbed by aging.

Muscular Dystrophy MDs are characterized by muscle fragility and weakness. Duchenne muscular dystrophy (DMD) is the most common form of MD. DMD patients show early signs of muscle H&E

weakness during childhood that progressively worsens and leads to an inability to walk and premature death around 20 to 30 years of age (9). DMD is caused by a genetic mutation that results in the absence of dystrophin, a structural protein involved in the interaction of the muscle fiber with the ECM. In absence of dystrophin, muscle fibers are fragile and prone to injury leading to continuous cycles of degeneration and regeneration (273). The chronically degenerative environment in MDs has negative effects on satellite cell function. In dystrophic muscles, satellite cells are constantly exposed to an activating environment. It has been speculated that this continuous activation results in premature exhaustion of the satellite cell pool. Primary cultures of myoblasts from dystrophic muscles show a much lower proliferative potential in vitro than healthy myoblasts, a phenomenon that is accentuated by aging (314). Moreover, it has been demonstrated that telomeres are significantly shorter in dystrophic satellite cells and it has been speculated that this phenomenon could be responsible for the decreased regenerative ability of muscle satellite cell observed in MDs (83, 256). Contradicting these ideas, biopsies from humans MD patients or dystrophic mice are characterized by elevated numbers of satellite cells even in advanced stages of dystrophy (52, 161, 243). In dystrophic muscles, satellite cells are surrounded by an altered microenvironment composed of fibrotic tissue, fat, and inflammatory cells (Fig. 18). Under dystrophic conditions injured myofibers appear to send cues to FAPs that favor adipogenesis and fibrosis (143, 303). A reduction of fibrosis in dystrophin-null mice (mdx) mediated by the application of TGF-β inhibitors decreases myofiber necrosis and improves muscle regeneration (66, 293). Similarly, inflammatory cells are deregulated in dystrophic muscles and impair muscle regeneration and satellite cell function. The chronic degenerative environment encourages the polarization of leukocytes, such as macrophages, toward the proinflammatory phenotype (M1) (307). Thus, anti-inflammatory corticosteroids remain one of the few therapeutic options that can temporarily ameliorate the symptoms of MDs (191).

Sirius red

Trichrome masson

Figure 18

Degeneration of dystrophic muscles. Images from dystrophic tibialis anterior muscles. H&E staining showing centronucleated fibers that indicate muscle regeneration. Sirius red (fibrotic tissue in red) and trichrome masson (fibrotic tissue in blue) illustrate the massive fibrosis response in the dystrophic muscles.

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Next to the altered extracellular environment, certain evidence suggests that intrinsic changes can affect satellite cell function in muscle dystrophy. Dystrophin stabilizes cells by binding to the dystroglycan (DAG) complex on the cell membrane surface. The DAG also serves as anchoring protein for ECM proteins such as laminin. In absence of dystrophin the DAG complex is strongly perturbed. Interestingly, it was found that disruption of DAG1 specifically in the myofibers (e.g., not in the satellite cells) results in a remarkably mild dystrophic phenotype (65). These results suggest that myofiber fragility is not the only problem underlying MD and that satellite cell dysfunction could play a role in the pathogenesis of this group of diseases. Overall, there are many conflicting results in the literature with regard to the role of satellite cells in MDs. One reason underlying this lack of consensus is the absence of an experimental model that appropriately mimics human DMD. Deletion of dystrophin gene in mdx mice does not result in a phenotype that is similar to the one observed in humans. Mdx mice appear to be able to compensate much more efficiently for the absence of dystrophin than humans and other higher organisms. It was hypothesized that this higher resistance to muscle degeneration could be mediated by compensation by a related structural protein such as utrophin or by a higher regenerative potential of mouse satellite cells (318).

Muscle Stem Cell Regenerative Therapies The myogenic potential of satellite cells can be perturbed by different pathologies and conditions. Research on regenerative therapies for skeletal muscle often focuses on delivering healthy myogenic cells into diseased muscles or investigates the possibility to improve or restore the endogenous myogenic potential of satellite cells. Muscle satellite cell transplantation has been studied extensively in the last decades and holds great therapeutic potential for diseases such as MD. In addition to restoring the satellite cell pool and to repairing the host myofibers, healthy myogenic donor cells can serve as vectors to mediate expression of normal alleles in muscle fibers they fuse to (Fig. 19) (228). Thus, fusion of healthy transplanted donor cells to dystrophic myofibers will allow the donor nuclei to express the normal dystrophin allele in the host muscle fibers. Unfortunately, such therapeutic approaches have many technical issues. There is for instance a need for immunosuppression, donor cells often show poor survival after injection and a high number of injections are required to get an effect on a given muscle. Moreover, the engraftment potential of in vitro cultured myoblasts is very low and the surviving myoblasts rapidly fuse to the fibers and show no long-term engraftment into the stem cell compartment (136). In contrast to in vitro cultured myoblasts it has been shown that freshly isolated satellite cells (especially the satellite stem cell subpopulation) are capable of long-term engraftment and can sustain multiple round of injuries (51, 67, 163, 255). However, obtaining

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sufficient numbers of freshly isolated satellite cells to treat multiple muscles is impractical. Thus, so far the success of myogenic cell therapy in humans is very limited (297). Niche components such as ECM proteins involved in selfrenewal (e.g., collagen VI or fibronectin) have been used to mimic the satellite cell microenvironment in vitro and can lead to improved transplantation efficiency of cultured cells (26, 110, 304). It was also shown that it is feasible to isolate viable satellite cells from postmortem tissue, which may allow for obtaining sufficient numbers of cells for therapy. Another source for myogenic cells could be the conditional expression of Pax7 in embryonic stem cells or inducible pluripotent stem cells. Myogenic cells generated with this technique can engraft in the satellite cell niche and contribute to muscle fibers (79). Myogenic cells have also been coinjected with growth factors or with other cell types that improve cell survival, migration, and engraftment (20, 38, 164, 173). For instance, short ex vivo treatment of satellite cells with Wnt7a considerably increases dispersion and engraftment that ultimately results in improved function of dystrophic muscles (23). However, despite these various improvements, myogenic cell transplantation is still far from being an efficient clinical therapy. An option that allows for circumventing the huge amount of intramuscular injections needed for transplantation (25-100 injections/cm3 ) is to use cell types that have the potential to be delivered systemically and migrate through the vasculature to engraft as satellite cells (280). Mesoangioblasts, CD133+, SP cells and others have shown potential for systemic delivery. However, the engraftment potential of these cell types into the satellite cell compartment appears to be limited (15, 85, 123, 199, 260). A promising avenue to improve muscle regeneration in disease is to boost the endogenous myogenic potential of satellite cells. Intramuscular injection of Wnt7a can increase satellite cell numbers, myofiber size and muscle force in mdx muscles (310). Pharmacological inhibition of p38 MAPK could be another strategy to rejuvenate old satellite cells (28, 74). Inhibitors of p38 are already investigated in different clinical trials for various inflammatory conditions (22). Similarly, JAK/STAT inhibitors were also demonstrated to restore old muscle satellite cell regenerative potential (237). However, administration of systemic inhibitors strongly increases the chances of adverse side effects.

Conclusion In this review, we summarized the characteristics of satellite cells and their functions in healthy and regenerating skeletal muscle. Especially, we showed that during muscle regeneration satellite cell fate is tightly regulated by intrinsic and extrinsic factors, a fragile balance that can be perturbed by diseases or degenerative conditions. Moreover, the relatively recent discovery of satellite cell subpopulations with selfrenewal potential greatly improved our understanding of the

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Satellite cellderived myogenic cells in culture


Freshly isolated satellite cells Donor muscle tissue

Injection of donor cells

Host skeletal muscle

Mosaic muscle

Host muscle fiber

Donor cells fusing to host fibers Genetically corrected host fiber

Donor nucleus

Figure 19 Satellite cell therapy. Schematic illustrating the different steps of cell therapy. Healthy satellite cells are isolated from donor muscle tissue and are cultured in vitro or directly transplanted into diseased host muscle. Healthy satellite cells will then fuse to host myofibers to form hybrid fibers. Addition of new healthy nuclei to host myofibers can partially correct genetic disorders such as muscular dystrophies. However, this therapeutic avenue has major technical limitations such as short-term engraftment, limited availability of donor cells, and poor cell migration and survival.

mechanisms that control the overall satellite cell pool. Future advances in molecular and genetic technologies will help unravel the mechanisms controlling these stem cells and will hopefully allow for the development of efficient therapies for muscle diseases.

Acknowledgements The studies from the laboratory of M. A. Rudnicki were carried out with support of grants from the Canadian

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Institutes for Health Research (MOP-81288, MOP-12080, and ERA-132935), the US National Institutes for Health (R01AR044031), the Muscular Dystrophy Association, Muscular Dystrophy Canada, the Stem Cell Network, and the Ontario Ministry of Economic Development and Innovation, and the Canada Research Chair Program. M. A. Rudnicki holds a Canada Research Chair in Molecular genetics. N. A. Dumont is supported by a Postdoctoral Fellowship from the Canadian Institutes for Health Research. We thank Caroline Brun for reviewing the manuscript.

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1059

Satellite cells and muscle regeneration in diseased human skeletal muscles.

Satellite cells in human skeletal muscle plasticity.

Characteristics of the Localization of Connexin 43 in Satellite Cells during Skeletal Muscle Regeneration In Vivo.

Brain and muscle Arnt-like 1 promotes skeletal muscle regeneration through satellite cell expansion.

Muscle Regeneration after Critical Illness: Are Satellite Cells the Answer?

Gαi2 signaling is required for skeletal muscle growth, regeneration, and satellite cell proliferation and differentiation.

Skeletal muscle satellite cells appear during late chicken embryogenesis.

Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity.

Lkb1 is indispensable for skeletal muscle development, regeneration, and satellite cell homeostasis.

BRE facilitates skeletal muscle regeneration by promoting satellite cell motility and differentiation.

Human Satellite Cell Transplantation and Regeneration from Diverse Skeletal Muscles.

Stem cells for skeletal muscle regeneration: therapeutic potential and roadblocks.

Skeletal muscle satellite cell diversity: satellite cells form fibers of different types in cell culture.

The PERK arm of the unfolded protein response regulates satellite cell-mediated skeletal muscle regeneration.

Towards understanding skeletal muscle regeneration.

Glycolysis in skeletal muscle regeneration.

TAK1 modulates satellite stem cell homeostasis and skeletal muscle repair.

STAT3 signaling controls satellite cell expansion and skeletal muscle repair.

AMP-activated protein kinase stimulates Warburg-like glycolysis and activation of satellite cells during muscle regeneration.

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

Skeletal myogenic differentiation of human urine-derived cells as a potential source for skeletal muscle regeneration.

Cellular players in skeletal muscle regeneration.

Adhesion in skeletal muscle during regeneration.

Differentiation capacities of skeletal muscle satellite cells in Lantang and Landrace piglets.