Adult
skeletal muscle has a remarkable ability to regenerate following
myotrauma. Because adult myofibers are terminally differentiated, the
regeneration of skeletal muscle is largely dependent on a small
population of resident cells termed satellite cells. Although this
population of cells was identified 40 years ago, little is known
regarding the molecular phenotype or regulation of the satellite cell.
The use of cell culture techniques and transgenic animal models has
improved our understanding of this unique cell population; however, the
capacity and potential of these cells remain ill-defined. This review
will highlight the origin and unique markers of the satellite cell
population, the regulation by growth factors, and the response to
physiological and pathological stimuli. We conclude by highlighting the
potential therapeutic uses of satellite cells and identifying future
research goals for the study of satellite cell biology.
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INTRODUCTION |
SKELETAL MUSCLES OF ADULT mammalian
species exhibit a remarkable capacity to adapt to physiological demands
such as growth, training, and injury. The processes by which these
adaptations occur are largely attributed to a small population of cells
that are resident in adult skeletal muscle and are referred to as
satellite cells. After their initial identification in 1961 (98), Mauro (117) described a cell closely
associated with the periphery of the frog myofiber and termed it a
satellite cell based on its location. Quiescent satellite cells are
physically distinct from the adult myofiber as they reside in
indentations between the sarcolemma and the basal lamina
(130). Adult skeletal muscle fibers are terminally
differentiated such that muscle growth and regeneration are
accomplished by satellite cells. In the unperturbed state, these cells
remain in a nonproliferative, quiescent state. However, in response to
stimuli such as myotrauma, satellite cells become activated,
proliferate, and express myogenic markers (satellite cells expressing
myogenic markers are also termed myoblasts). Ultimately, these cells
fuse to existing muscle fibers or fuse together to form new myofibers
during regeneration of damaged skeletal muscle (12, 167).
Since the original description of the myogenic satellite cell,
considerable interest and research efforts have focused on myogenic
satellite cell biology. These research efforts have enhanced our
understanding of muscle growth, remodeling, and regeneration. In
addition, new paradigms have been proposed regarding the regenerative capacity and the plasticity of the myogenic satellite cell population (108, 119, 139, 168, 169). These paradigms suggest that
the satellite cell population not only has a remarkable capacity for muscle regeneration but may also contribute to alternative muscle and
nonmuscle lineages and may have clinical applications in the treatment
of devastating and deadly diseases such as muscular dystrophy.
The current review attempts to integrate the anatomic, physiological,
biochemical, and molecular properties that regulate the myogenic
satellite cell population. We begin with a brief overview of vertebrate
myogenesis, highlighting the populations of myoblast precursor cells
that contribute to muscle development, and outline a well-described
genetic hierarchy that is important in muscle specification. We
describe the limited gene expression profile and the distinguishing
morphological characteristics of the satellite cell population. We
describe the inductive signals that regulate the satellite cell in
vitro and in well-described physiological models. Finally, we will
review the stem cell-like features of the satellite cells with emphasis
on the novel strategies that may be pursued in the future for the
treatment of debilitating myopathies.
Importantly, myogenic satellite cell biology remains an emerging
field of scientific inquiry, such that satisfying and complete answers
to the most fundamental questions are currently unavailable. In this
review, we will provide a summary of the current knowledge in this area
and highlight fertile areas for future research.
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BUILDING OF MUSCLE WITH WAVES OF PRECURSOR CELLS |
Anatomic and molecular mechanisms during muscle regeneration have
been postulated to recapitulate muscle development. Although current
evidence suggests the regenerative process may be more complex, an
understanding of muscle development is important to appreciate the
anatomic and molecular network associated with muscle regeneration.
During embryogenesis, the head, trunk, and limb skeletal muscles
develop as separate lineages. Of the three germ layers in the early
embryo, the paraxial mesoderm gives rise to the somite (Fig.
1A). The somite is subdivided
into the dorsomedial (epaxial) domain, which generates the muscles of
the back, and the ventrolateral (hypaxial) domain, which gives rise to
the abdominal, intercostal, and limb musculature (see Refs.
123 and 140 for review).
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Fig. 1.
Derivation of muscle precursor cells during mouse embryogenesis.
A: precursor cells from the epaxial region of the somite
migrate to form the back musculature. Precursor cells from the hypaxial
region of the somite migrate to the newly formed limb buds. Note the
migrating limb precursor cells form dorsal and ventral masses, which
will later become the extensor and flexor muscle groups of the limb.
Surrounding structures such as the neural tube, notochord, dorsal
aorta, and overlying ectoderm potentially provide signals regulating
precursor cell movements and fate. B: members of the MyoD
family play an integral role in skeletal muscle myogenesis. MyoD and
myf5 expression is involved in determination of
precursor cells to a myogenic fate, whereas myogenin and MRF4
expression is associated with terminal differentiation.
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During somitogenesis, cross talk involving growth factors (Wnt
proteins, Sonic hedge hog, bone morphogenic proteins, and so forth) and
transcription factors (myf5, MyoD, Pax-3, and so forth) occurs between the developing somite and the anatomically adjacent structures, including the overlying ectoderm, the ventromedial neurotube and notochord, and the vascular structures including the
aorta (38, 113, 123, 131, 145). The constellation of these
positional cues (i.e., growth and transcription factors) results in the
specification of muscle through the regulation of a distinct molecular
(hierarchical) cascade.
Since the discovery of MyoD in 1987, the role of the myogenic
basic helix-loop-helix (bHLH) transcription factors in skeletal myogenesis has been defined in elegant detail by several groups (19, 126, 137, 147, 148, 192, 200, 207). This subset of
the bHLH family includes MyoD, myf5, myogenin, and
MRF4. Each of these myogenic bHLH proteins forms heterodimeric DNA
binding complexes that include other bHLH proteins of the E2 gene
family (E12 and E47) and bind a canonical DNA sequence, CANNTG (E-box), within enhancer elements of genes that encode terminal differentiation markers of the skeletal muscle lineage (41, 103). MyoD
family members share the ability to activate skeletal muscle
differentiation when expressed ectopically in nonmuscle cells
(115, 193). The essential role played by bHLH proteins in
skeletal myogenesis has been demonstrated unambiguously by gene
disruption experiments (78, 138, 142, 147, 148, 157, 185).
The results of these gene knockout experiments support a role for
myf5 and MyoD in the determination of the myogenic cell
fate and the formation of myoblasts during embryogenesis (Fig.
1B). Myogenin and MRF4 appear to function in activation of
muscle differentiation (149, 175). Although a number of
biochemical and transgenic studies support an integral role for the
myogenic bHLH proteins during development, it is also clear that the
MyoD family members interact in a combinatorial fashion with known
transcription factors such as members of the MADS box family (i.e.,
myocyte enhancer factor 2 or MEF-2; Refs. 41,
103), cell cycle regulatory proteins, and currently
undefined factors to regulate myogenesis (see Refs. 126
and 175 for review). Although the role of MyoD family members during embryogenesis has been defined in great detail, the functional role of these family members in established postnatal skeletal muscle
remains unclear. Studies support the localization of MyoD and myogenin
in fast-twitch and slow-twitch adult myofibers, respectively, suggesting a function for these myogenic regulatory factors in fiber-specific contractile protein gene expression (84, 85, 171). Future studies utilizing transgenic technologies such as conditional or tissue-restricted knockout strategies of these myogenic regulatory factors will be useful in the definition
of their role in adult skeletal muscle.
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MUSCLE PRECURSOR CELLS AND LIMB DEVELOPMENT |
Whereas the cells associated with the epaxial region of the somite
contribute to the primary myocytes of the myotome, the hypaxial region
of the somite (at the level of the limb) contributes to the migratory
limb muscle precursor cells (141). Specification, migration, and differentiation of the myogenic precursor cells to their
distal targets are complex processes involving intrinsic and extrinsic
cues of which little is known. Recent studies have shown that these
myogenic precursor cells express the paired domain transcription factor
Pax-3 (181), the tyrosine kinase receptor c-Met
(205), the homeodomain transcriptional repressor msx1
(11, 189), and the homeodomain transcription factor Lbx 1 (17, 71) but lack expression of the myogenic regulatory
factors of the MyoD family. After migration to the developing limb,
these precursor cells coalesce into the dorsal and ventral premuscle
masses, which will be the future flexor and extensor compartments of
the forelimb and now express members of the MyoD family (19, 137,
141).
Targeted gene disruption studies (i.e., knockout mouse models) have
begun to provide insight into the molecular regulation of limb
development. In mice lacking either Pax-3 (16, 60) or
c-Met (50), limb precursor cells fail to migrate into the limb, resulting in the complete loss of limb muscles. Combinatorial knockout experiments crossing the Pax-3 mutant mouse
(Splotch) into the myf5 null background
result in a further perturbation of myogenesis and an absence of both
limb and body wall muscle (181). Additional studies
support a genetic hierarchy where Pax-3 mediates the activation of
other myogenic regulatory factors (myf5 and MyoD) and
functions as a key regulator of somitic myogenesis (113,
181). In addition, Pax-3 functions in the specification of the
limb precursor cells and is upstream of both c-Met and Lbx 1 (17,
50, 60, 181). Inactivation of the Lbx 1 locus by homologous
recombination results in an extensive loss of limb muscles, although
residual muscle groups are still present. This finding suggests that
Lbx 1 is not required for the specification of limb muscles but may
function in the determination of which migratory highway the precursors
should pursue (17, 71).
Proliferating limb myoblasts coalesce into the ventral and dorsal
premuscle masses, withdraw from the cell cycle, and form multinucleated
primary myofibers at approximately embryonic day 13 postcoitum (E13) in the mouse embryo. In a process that is less clearly
defined, secondary myofibers form parallel to the primary fibers and
constitute the predominant multinucleated myofibers during the latter
stages of embryogenesis (E15-E16 in the mouse) and in postnatal
skeletal muscle (56, 123). Studies suggest that two
distinct lineages generate primary myofibers (i.e., embryonic myoblasts) and the secondary myofibers (i.e., fetal myoblasts). Furthermore, primary myofibers differ from secondary myofibers in their
temporal development, size, number, and expression of myosin
heavy-chain isoforms (123, 175).
The discovery of satellite cells in the early 1960s demonstrated the
existence of yet an additional population of proliferative cells that
contributed to postnatal growth, the maintenance of adult skeletal
muscle, and the repair of damaged myofibers. Myogenic satellite cells
are present in the limbs of midgestational mouse embryos after E15
(35). After birth, the satellite cell population accounts
for ~30% of sublaminar muscle nuclei in neonatal hindlimb skeletal
muscle (12). These neonatal satellite cells fuse to growing myofibers to contribute additional nuclei during postnatal growth of skeletal muscle.
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SOMITIC VS. NONSOMITIC ORIGIN OF SATELLITE CELLS |
Previous studies support the hypothesis that muscle precursor
cells, including the myogenic satellite cell population, originate from
the multipotential mesodermal cells of the somite (58, 141,
167). The support for this hypothesis is primarily derived from
chimeric or interspecies grafting experiments that have been performed
in avian models. These fate-mapping studies involved the
transplantation (or exchange) of embryonic somites from donor quail
embryos into host chick embryos (27, 105). The
transplanted quail cells have distinguishing morphological
characteristics and were observed to migrate from the somite and
contribute to both the limb muscles and the satellite cell population
in postnatal chick skeletal muscle. Satellite cells have been isolated
from the fetal skeletal muscle from an E15 or older mouse embryo,
suggesting that satellite cells populate the developing limb during the
latter stages of embryogenesis (34, 35, 36). Whether the
satellite cells migrate from the somite as a distinct lineage or
whether they originate from a preexisting lineage (i.e., embryonic or fetal myoblasts) in the developing limb is unclear. Nevertheless, the
underlying concept was that each of the myoblast precursor cells (i.e.,
embryonic myoblasts, fetal myoblasts, and satellite cells) was a
derivative of the somite.
This paradigm has recently been challenged, as studies have suggested
that multipotential cells of nonsomitic origin may be the precursors of
the satellite cell (45, 139). De Angelis et al.
(45) reported that cells isolated from the embryonic dorsal aorta had a similar morphological appearance and a similar profile of gene expression to that of satellite cells. Furthermore, transplantation of the aorta-derived myogenic cells into newborn mice
revealed that this cell population participated in postnatal muscle
growth, regeneration, and fusion with resident satellite cells. The
authors proposed that satellite cells may be derived from endothelial
cells or a precursor common to both the satellite cell and the
endothelial cell.
Derivation of the satellite cell from the somite or a nonsomitic source
need not be mutually exclusive. Conceivably, both lineages may
contribute under physiological or pathological states to the satellite
cell population. Additional fate-mapping strategies will be needed to
further define and dissect the lineage derivation of all the satellite
cells that reside in adult skeletal muscle. These studies will be
important in defining whether there is a common lineage source for the
entire satellite cell population and a common lineage source for cells
that have regenerative capacities in both muscle and nonmuscle tissues.
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SATELLITE CELL IDENTIFICATION |
Anatomic identification.
Resident within adult skeletal muscle is a pool of undifferentiated
mononuclear cells termed satellite cells because of their anatomic
location at the periphery of the mature, multinucleated myotube. The
defining characteristic of the satellite cell is that the basal lamina
that surrounds the satellite cell and the associated myofiber is
continuous (167). As shown in Fig.
2, the identification of this cell
population has historically utilized ultrastructural techniques
(66, 161). Other distinguishing morphological features of
the satellite cell population include a relatively high
nuclear-to-cytoplasmic ratio with few organelles, a smaller nuclear
size compared with the adjacent nucleus of the myotube, and an increase
in the amount of nuclear heterochromatin compared with that of the
myonucleus (167). These morphological features are
consistent with the finding that satellite cells are relatively
quiescent and transcriptionally less active. These distinguishing
features are absent following activation or proliferation of the
satellite cells in response to growth, remodeling, or muscle injury.
After activation, the satellite cells are more easily identified as
they appear as a swelling on the myofiber with cytoplasmic processes
that extend from one or both poles of the cell (Fig. 2; Ref.
167). Associated with the increase in mitotic activity, there is a reduction in heterochromatin, an increase in
cytoplasmic-to-nuclear ratio, and an increase in the number of
intracellular organelles (167).
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Fig. 2.
Satellite cells occupy a sublaminar position in adult skeletal
muscle. In the uninjured muscle fiber, the satellite cell is quiescent
and rests in an indentation in the adult muscle fiber. The satellite
cells can be distinguished from the myonuclei by a surrounding basal
lamina and more abundant heterochromatin. When the fiber becomes
injured, the satellite cells become activated and increase their
cytoplasmic content. The cytoplasmic processes allow for chemotaxis of
the satellite cell along the myofiber. Bar = 1 µm.
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Satellite cell markers.
The profile of gene expression of the quiescent satellite cell as well
as their activated and proliferating progeny is largely unknown. The
quiescent satellite cells do not express myogenic regulatory factors of
the MyoD or MEF2 families or other known markers of terminal
differentiation (33, 119, 201). Through the identification
of satellite cell markers, biologists will be able to address issues
related to the developmental origin of the satellite cell, the cell
cycle control, and the molecular regulation of this unique cell
population during growth and regeneration. Several satellite cell
markers have been identified and are restricted to either the
quiescent, activated, or proliferative state or are expressed more
broadly (Table 1).
We have previously determined that myocyte nuclear factor (MNF or
Foxk1), a member of the winged helix transcription factor family, is
localized to the quiescent satellite cell in adult skeletal muscle
(64). We have identified two alternatively spliced isoforms for MNF and termed them MNF-
and MNF-
(64, 203, 204). These two alternatively spliced isoforms are reciprocally expressed during myogenesis and during muscle regeneration, suggesting that the two isoforms of MNF may exert opposing effects on target genes
at discrete steps during muscle regeneration or in renewal of the
satellite cell population (63). Using a RT-PCR assay, we
have shown that MNF- is the principal form expressed in quiescent satellite cells, whereas MNF- predominates in proliferating
satellite cells following muscle injury. Disruption of the MNF locus,
mutating both isoforms, resulted in a severe growth deficit, a marked
impairment in muscle regeneration (63), and a decreased
number of satellite cells in adult MNF mutant skeletal muscle (Hawke
and Garry, unpublished observations). Additional winged helix family
members have also been identified in stem cells or regenerating cells
including Genesis (Foxd3; Ref. 83), which is expressed
selectively in embryonic stem cells, and a protein related to
hepatocyte nuclear factor-3 (HNF3/forkhead homolog 11), which
has been identified in regenerating hepatocytes (206).
Utilizing single cell RT-PCR analysis, Cornelison and Wold
(33) characterized the satellite cells as a heterogeneous
population based on their profile of gene expression. Additionally,
they identified c-Met, the receptor for hepatocyte growth factor (HGF), as a marker of quiescent satellite cells. HGF is a potent mitogen for
satellite cells and has been shown to be important in the migration of
the myogenic precursor cells from the somite to the developing limb
(5, 14). Moreover, c-Met deficient embryos fail to form
limb skeletal muscle due to a lack of myogenic precursor cells
(14, 112).
Irintchev and colleagues (87) identified M-cadherin, a
calcium-dependent cell adhesion molecule, as a unique marker of the satellite cell pool. M-cadherin is only expressed in a subpopulation of
the quiescent cell pool; however, its expression is increased when the
satellite cells become activated in response to a stimulus (9,
33). Recent studies suggest that other cell adhesion molecules,
neural cell adhesion molecule (NCAM) and vascular adhesion molecule-1 (VCAM-1), are also potential markers of quiescent satellite cells (39, 89). The role of these adhesion molecules is
unclear but collectively (NCAM, VCAM-1, and M-cadherin) may function in the adhesion of the satellite cell to the basal lamina of the myofiber
and may participate in the migratory capacity of this cell population
in response to stimuli. NCAM is expressed in both myofibers and
satellite cells, whereas VCAM-1 is broadly expressed during
embryogenesis but limited to satellite cells in adult muscle (39). Furthermore, VCAM-1 has been shown to mediate
satellite cell interaction with leukocytes following injury
(89).
Recent work from the Rudnicki laboratory (169) identified
the paired box transcription factor, Pax7, expressed selectively in
quiescent and proliferating satellite cells. Analysis of the Pax7
mutant skeletal muscle revealed a complete absence of satellite cells.
This novel finding supports the hypothesis that Pax7 is essential for
the specification of the satellite cell population. Future studies will
be important in the definition of Pax7 downstream target genes in the
satellite cell population and may provide insight into the regulation
of this cellular pool.
Identification of other satellite cell markers is the focus of current
research efforts. Emerging candidates include the cell surface antigen
Sca-1 (stem cell antigen-1; Refs. 88, 177), the glycoprotein Leu-19 (94, 162), the anti-apoptotic
factor Bcl-2 (106, 123), CD34 (9), and
interferon regulatory factor-2, which is a transcription factor that
mediates VCAM-1 expression in skeletal muscle (89).
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SATELLITE CELL QUANTITATION AND DISTRIBUTION |
Quantitation of the satellite cell population in adult skeletal
muscle has been possible primarily through the use of ultrastructural techniques. More recently, immunohistochemical techniques have been
utilized for the identification of the satellite cell pool. Although a
limited number of markers for the quiescent satellite cell population
exist, proliferating satellite cells, as measured by
[3H]thymidine or bromodeoxyuridine (BrdU) incorporation,
can be identified immunohistochemically for the coexpression of the
MyoD family members or the intermediate filament protein, desmin
(15, 104, 123). MyoD expression occurs early during the
activation of the satellite cell population (within 6 h following
muscle injury) (63, 74, 100, 119, 134). In addition,
several nonselective markers of cellular proliferation have been used
to characterize the proliferating satellite cell pool. These markers of
cellular proliferation include proliferating cell nuclear antigen
(90), BrdU (163), and
[3H]thymidine (128).
Satellite cell number is dependent on the species, age, and muscle
fiber type (Table 2; see Ref.
167 for review). Satellite cells constitute ~30% of the
muscle nuclei in the neonate and decrease with age to ~4% in the
adult and ~2% in the senile (29-30 mo) mouse
(176). The decrease in the percentage of satellite cells
with aging (i.e., senile rodent) is the result of an increase in
myonuclei (oxidative and glycolytic myofibers) and a decrease in total
number of satellite cells (glycolytic myofibers) (12, 66,
167).
The satellite cell distribution between muscle groups is a result of
the heterogeneity in satellite cell content between muscle fiber types.
An increase in satellite cell density has been demonstrated in
association with the proximity of capillaries (161),
myonuclei (18, 161), and motoneuron junctions
(199). The proximity of satellite cells to these anatomic
structures suggests a permissive role in the regulation of the cellular
pool. In support of this hypothesis, oxidative fibers (characterized by
increased capillary and motoneuron density compared with glycolytic
myofibers) demonstrate a five to six times greater satellite cell
content (65, 161).
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GROWTH FACTORS AS REGULATORS OF THE SATELLITE CELL POPULATION |
The process of muscle regeneration requires the influence of
growth factors and a sequence of cellular events, which results in the
regulation of the satellite cell population (Table
3, Fig.
3; Refs. 73,
168). Many of the studies that have examined the effect of
growth factors on satellite cell biology have utilized satellite cell
cultures. These studies have defined the effect of growth factors alone
or in combination and have provided valuable insight into the
regulation of the satellite cell. Admittedly, in vitro studies are
limited due to the lack of permissive and repressive factors that are
present in vivo and may influence cellular activity. Currently, most
satellite cell cultures are derived from neonatal skeletal muscle due
to the abundance of satellite cells in these tissues compared with
older animals (>30% in young animals vs. ~5% in older animals;
Ref. 143). The population and age of the satellite cell is
an important consideration in cell culture preparations as the response
of aged, quiescent satellite cells to growth factor stimulation differs
compared with young, proliferating satellite cells (182).
This section will provide a brief introduction of growth factors that
are important in the regulation of satellite cell proliferation,
differentiation, and motility (for review, see Table 3).
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Fig. 3.
Factors modulate satellite cell activity. Growth factors and
hormones are released from a number of tissues and modulate satellite
cell activity (chemotaxis, proliferation, and differentiation). These
factors utilize signaling pathways in the regulation of the satellite
cell. LIF, leukemia inhibitory factor; IL-6, interleukin-6. EGF, FGF,
HGF, IGF, PDGF, and TGF, endothelial-derived, fibroblast, hepatocyte,
insulin-like, platelet-derived, and transforming growth factors,
respectively.
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Insulin-like growth factors.
Skeletal muscle secretes insulin-like growth factors I and II (IGF-I
and IGF-II), which are known to be important in the regulation of
insulin metabolism (3, 109, 186). In addition, these
growth factors are important in the regulation of skeletal muscle
regeneration. IGF-I and IGF-II increase satellite cell proliferation
and differentiation in vitro (Table 3). The importance of these growth
factors was demonstrated with the intramuscular administration of IGF-I
into older, injured animals. In this study, IGF-I administration (using an osmotic minipump) resulted in enhanced satellite cell proliferation and increased muscle mass (26). Moreover, skeletal muscle
overload or eccentric exercise results in elevated IGF-I levels,
increased DNA content (suggesting an increase in satellite cell
proliferation), and a compensatory hypertrophy of skeletal muscle
(1, 202).
IGF-I appears to utilize multiple signaling pathways in the regulation
of the satellite cell pool. The calcineurin/NFAT, mitogen-activated protein (MAP) kinase, and phosphatidylinositol-3-OH kinase (PI-3K) pathways have all been implicated in satellite cell proliferation (25, 32, 170). IGF-I-stimulated satellite cell
differentiation appears to be mediated through the PI-3K pathway
(32). Additional studies, utilizing genetic mouse models
(i.e., transgenic overexpression or knockout models), may further
define the regulation and signaling pathways of the IGFs and satellite
cell biology (8, 25, 132).
Hepatocyte growth factor.
Hepatocyte growth factor (HGF) is a multifunctional cytokine initially
described as a mitogen in mature hepatocytes (122). Recently, HGF and its receptor c-Met have been localized to satellite cells and adjacent myofibers but are absent in the adjacent
fibroblasts. In addition, HGF expression is proportional to the degree
of muscle injury (33, 174, 182). Multiple roles for HGF
have been proposed for the regulation of the satellite cell, including
a role as a potent chemotactic factor, an activator of the satellite
cell, and an inhibitor of myoblast differentiation (Table 3). HGF is capable of activating and selectively promoting satellite cell proliferation (4). Furthermore, HGF administration
attenuates satellite cell differentiation through the transcriptional
inhibition of the myogenic regulatory factors (i.e., MyoD and myogenin)
(62).
Fibroblast growth factors.
Fibroblast growth factor (FGF) has nine different isoforms (FGF-1 to
FGF-9). Although many of the FGF isoforms are broadly expressed, FGF-6
is restricted to skeletal muscle (59). Sheehan and Allen
(173) investigated in detail the role of the FGF family on
satellite cell proliferation in culture. In these studies, it was
demonstrated that FGF-1, -2, -4, -6, and -9 stimulated cellular
proliferation, whereas FGF-5, -7, and -8 had no mitogenic activity. The
investigators further observed that addition of HGF to either FGF-2,
-4, -6, or -9 resulted in a synergistic increase in satellite cell
proliferation. In addition to an increase in satellite cell
proliferation, the FGF family has also been observed to attenuate
satellite cell differentiation to myofibers (30, 91, 173,
178).
Floss et al. (59) reported that mice deficient for FGF-6
(i.e., knockout mice at the FGF-6 locus) have impaired satellite cell
proliferation and a subsequent defect in muscle regeneration in
response to a crush injury. In contrast, Fiore et al. (57) pursued a similar targeting strategy to mutate the FGF-6 locus and
observed an absence of defects in response to either a crush injury or
chemically induced injury (notexin). Consequently, the functional
role(s) of FGF-6 during muscle regeneration remains unclear.
Nevertheless, these studies underscore the ability of redundant family
members to compensate for one another and result in preserved function
under pathological conditions (i.e., mouse knockout models).
The release of FGF-2 from the damaged myofibers, like HGF, is
proportional to the degree of injury (29). FGF levels are coordinated with FGF receptor expression. When receptor expression is
increased, satellite cells propagated in culture demonstrate an
increased proliferation and decreased differentiation
(160). Conversely, when receptor expression is diminished,
proliferation is decreased and there is a concomitant increase in
satellite cell differentiation. Interestingly, during the period of
satellite cell activation and proliferation (0-48 h after injury),
FGF receptor (FGF-R1) mRNA is increased fivefold, and this increase is
further enhanced in the presence of HGF (173).
The signaling pathway(s) that transduces the FGF signal has recently
been investigated with the use of both transgenic techniques and
pharmacological inhibitors (92). These studies revealed that the MAP kinase pathway is important in transducing the FGF-induced increase in satellite cell proliferation; however, the MAP kinase signaling pathway did not mediate the FGF-mediated repression of
satellite cell differentiation.
Transforming growth factors.
Transforming growth factor- (TGF-) is the prototypical family
member of cytokines that includes bone morphogenic protein and
growth-differentiation factors. The TGF- family of cytokines transduces their signal through the SMAD family of proteins (see Ref.
196 for review). Generally, the TGF- family members
function to inhibit muscle proliferation and differentiation (3,
70, 97, 99, 208) by silencing the transcriptional activation of
the MyoD family members (115). This inhibition of
differentiation by TGF-, like FGF, persists even in nonmuscle cell
lines engineered to ectopically express members of the MyoD family
(97, 115, 184). The combination of IGF-I or FGF with
TGF- was unable to alter the TGF--induced attenuation of
satellite cell differentiation; however, TGF- action had little
effect on IGF-I- or FGF-mediated increases in proliferation
(70). During muscle regeneration, TGF- receptor levels
(TGF- RII) and TGF ligand are reciprocally expressed, resulting in
the initial promotion of cellular proliferation followed by enhanced
muscle differentiation (159).
Interleukin-6 cytokines.
Leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) are members
of the IL-6 family of cytokines produced by many different cells,
including myoblasts and macrophages. These cytokines share a common
receptor component, and their actions are mediated through the same
signaling pathways (81, 144). Skeletal muscle regeneration after injury in LIF mutant mice is attenuated, whereas exogenous administration of LIF increased the regenerative process and produced enlarged myofibers (101). The permissive effect of LIF was
associated only with the muscle lineage and had no effect on nonmuscle
cells in skeletal muscle (101). IL-6 promotes the
degradation of necrotic tissue, synchronizes the cell cycle of
satellite cells, and induces apoptosis of macrophages following
muscle injury (22). Unlike LIF, however, IL-6 expression
in injured muscle does not increase satellite cell proliferation
(96). Collectively, this family of growth factors appears
to play an integral role in skeletal muscle regeneration.
Many other factors may be involved in the regulation of the satellite
cell in adult skeletal muscle. Nitric oxide, platelet-derived growth
factor, endothelial-derived growth factor, and testosterone have been
shown to mediate satellite cell activity (6, 93, 133).
Obviously, the regulation of satellite cells is orchestrated by
numerous factors in a temporal and concentration-dependent fashion
during regeneration.
Few animal studies have examined the effect of growth factors in vivo.
Chakravarthy et al. (26) observed that local IGF-I administration to atrophied muscle increased satellite cell
proliferation and muscle mass within 2 wk. Unlike the observations with
IGF-I, the intramuscular injection of HGF, at specified intervals
following skeletal muscle injury, increased satellite cell
proliferation and either had no effect or impaired the rate of
regeneration (124). Similarly, administration of FGF at
timed intervals and selected dosages did not appreciably affect muscle
regeneration (125). Future studies that combine cell
culture methodologies and overexpression or loss of function models
using molecular technologies will be helpful in the definition of the
role of growth factors in satellite cell biology.
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FUNCTIONAL RESPONSES OF SATELLITE CELLS TO PHYSIOLOGICAL
STIMULI |
Hypertrophic stimuli.
Load-induced hypertrophy (chronic stretch, agonist ablation, tenotomy)
and resistance training are physiological challenges that promote a
hypertrophic response in both human and animal models
(154-156, 197). Hypertrophic growth of skeletal
muscle is stimulated by short bursts of muscle activity against high
resistance. Resistance training induces muscle hypertrophy through a
process of satellite cell activation, proliferation, chemotaxis, and
fusion to existing myofibers to contribute to muscle growth (Fig.
4; Ref. 167). The migratory
capacity (chemotaxis) of satellite cells is dependent on the integrity
of the basal lamina. After the rupture or interruption of the basal
lamina in response to myotrauma, satellite cells may migrate to
adjacent myofibers utilizing tissue bridges (164, 191). In
response to limited myotrauma, where no rupture of the basal lamina
occurs, satellite cells migrate from the proximal intact portion of the
myofiber, under the basal lamina, to the site of injury to participate
in the repair process (165, 167).
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Fig. 4.
Satellite cell response to myotrauma. *Skeletal muscle trauma or
injury may be minor (e.g., resistance training) or may be more
extensive (e.g., toxin injection, Duchenne muscular dystrophy). In
response to an injury, satellite cells become activated and
proliferate. Some of the satellite cells will reestablish a quiescent
satellite cell pool through a process of self-renewal. Satellite cells
will migrate to the damaged region and, depending on the severity of
the injury, fuse to the existing myofiber or align and fuse to produce
a new myofiber. In the regenerated myofiber, the newly fused satellite
cell nuclei will initially be centralized but will later
migrate to assume a more peripheral location.
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Exercise-induced myotrauma initiates an immune response, resulting in
the influx of macrophages into the damaged region. After the acute
insult, macrophage infiltration peaks within 48 h
(186). Initially, the role of these blood-borne
macrophages was believed to be limited to phagocytosis and the
digestion of myonecrotic fibers. However, additional roles for
macrophages during the early stages of muscle repair are emerging.
Macrophages are essential in the orchestration of the repair process as
they secrete a collection of cytokine factors that regulate the
satellite cell pool (133). Importantly, in the absence of
a macrophage response, muscle regeneration is absent; in the presence
of an enhanced macrophage response, there is an increase in satellite
cell proliferation and differentiation (110).
In response to resistance training, myotrauma results in the release of
growth factors that will, in part, regulate the satellite cell
population during regeneration (Fig. 3 and Table 3). For example, IGF-I
is upregulated in response to hypertrophic signals in skeletal muscle
and promotes proliferation and fusion of the satellite cell pool
(1, 26, 186). As outlined in the previous section,
additional growth factors and/or cytokines including LIF and members of
the TGF- family may play a role in the signaling or commissioning of
the satellite cells to participate in the hypertrophic remodeling
response. Although a number of questions remain regarding the role of
the satellite cell in muscle remodeling, the primary physiological
consequence of the hypertrophic response is to produce a muscle with a
greater capacity for peak force generation.
Atrophic stimuli.
Atrophy of skeletal muscle results in a reduction in myonuclei number
and can be induced by numerous factors including denervation, hindlimb
suspension, and malnutrition (73). Atrophy and remodeling that result from muscle disuse can be produced in laboratory rodents physiologically by tail/hindlimb suspension or immobilization of
specific muscle groups in plaster casts or pathologically through denervation. The response of the satellite cells appears to be pleiotropic and dependent on the atrophic stimulus.
In adolescent rats, hindlimb suspension results in an irreversible
remodeling process, including decreased satellite cell content and an
impaired proliferative capacity within 3 days of unloading in both the
oxidative slow-twitch soleus and the fast-twitch extensor digitorum
longus (EDL) muscles (44, 129). Thus the atrophic stimulus
in the adolescent animal may irreversibly alter the developmental
program for myofibers to accrue nuclei even with the resumption of
weight bearing. A similar reduction in the satellite cell population
was observed in adult rat hindlimbs using an immobilization model as an
alternative atrophic stimulus (188). In contrast to
adolescent animals, remobilization of the hindlimb was accompanied by
increased myofiber regeneration, supporting the hypothesis that,
following completion of the developmental program, adult satellite
cells are capable of activation and proliferation to repopulate
atrophied muscle (129, 188).
Unlike the other forms of atrophy, denervation is a pathological rather
than physiological stimulus. Denervation produces a form of disuse
atrophy that includes myofiber degeneration and is accompanied by
distinctive changes in the myonuclei and quiescent satellite cells
(102, 111, 153). In the unperturbed condition, there are
an increased number of satellite cells associated with the
neuromuscular junction (199). It is conceivable that
neurotrophic factors are important in satellite cell homeostasis. A
number of laboratories have reported that the percentage of satellite cells increase (from 3 to 9%) during the initial period following denervation (118, 187). However, a prolonged period of
denervation results in a significant decrease in the percentage of
satellite cells (3 to 1% following an 18-mo period of denervation).
Viguie et al. (187) hypothesize that the progressive
decline in the satellite cell pool may be the result of satellite cell
apoptosis. Alternatively, denervation may result in a lack of
neurotrophic input (including growth factors) that negatively impacts
satellite cell function and content.
Long-term denervation has considerable clinical implications.
Denervation for periods of 6-18 mo results in the inability of
skeletal muscle to reestablish a preinjury functional capacity even if
neuronal sprouting and regeneration occurs (180). The mechanisms for this phenomenon are unclear, but considerable data support the conclusion that the intact neuromuscular junction and the
denervation model mediate positive and repressive influences, respectively, on the satellite cell pool (187).
Aging.
Recent advances have allowed biologists to interrogate the
proliferative history of a cellular population. With each cell replication, there is ~100 bp lost from the ends of eukaryotic chromosomes (47, 48, 151, 172). The ends of the
chromosomes contain TTAGGG repeats termed telomeres. The length of
these telomeres reflects the number of replications of a particular
cell and its proliferative capacity. Using this technology,
investigators now have the ability to analyze the proliferative history
and future capacity of the satellite cell, providing valuable insight
into the effects of aging and disease in this cell population.
As age progresses, there is an impairment of skeletal muscle
regeneration following injury (see Ref. 72 for review). A
decrease in satellite cell number and/or proliferative capacity has
been used to explain this phenomena. Support for this hypothesis is observed in rodent models, as increasing age is associated with a
decrease in satellite cell number and a reduced proliferative capacity
(52, 66, 166). In contrast to the rodent model, a decrease
in human satellite cell population and proliferative ability is
observed only during the childhood years. For example, neonatal
(5-day-old) and infant (5-mo-old) satellite cells are capable of ~60
and 45 replications, respectively, whereas 9-yr-old and 
60-yr-old
humans are both capable of 20-30 replications (151). However, as aging progresses, satellite cells (60-yr-old skeletal muscle) fuse to form thinner, more fragile myotubes (151).
Thus, despite a normal ability to proliferate, the satellite cells of older humans have a reduced capacity to repopulate the myofiber population.
The impaired regenerative response that is observed with aging in
humans thus appears to be much more complex than satellite cell
senescence alone. Results from cross-transplantation experiments suggest that the host environment is a critical factor in the ability
of older skeletal muscle to regenerate (23, 24). Cross transplantation of EDL muscles between 4-mo-old and 24-mo-old rats
demonstrated that mature skeletal muscle was as capable as young
skeletal muscle in recovering from transplant and from toxin-induced injury. However, young (4-mo-old) nerve-muscle autografts functioned significantly better than old (24-mo-old) nerve-muscle autografts, as
determined by the measurement of mass and maximum isometric force,
suggesting that the ability for neuronal regeneration may be a critical
factor in activating the satellite cell response and, ultimately,
regenerating the damaged muscle (23, 24).
Other factors within the host environment affect the efficiency of
skeletal muscle regeneration as aging progresses. A thickening of the
basal lamina (176), increased fibrosis within skeletal muscle (114), and reduced capillary density
(31) may also contribute to impaired regeneration.
Inflammatory factors (macrophages and associated cytokines) are
essential for the normal satellite cell response to injury. Aging
negatively impacts the immune response, resulting in a decrease of the
inflammatory factors and macrophages (43). In association
with an impaired immune response, there are reduced serum levels of
growth factors including IGF-I in aged rats and humans (150,
183). After multiple cycles of atrophy, there is no restoration
of muscle mass or satellite cell proliferation in aged rats even after
9 wk of recovery. However, local IGF-I administration to the atrophied
muscle resulted in significant increases in mass and satellite cell
proliferation within 2 wk (26).
An unresolved question with regard to satellite cells and the aging
process is whether repeated exhaustive and/or resistance training
exercise programs have a negative impact on the long-term satellite
cell content? This question is applicable to the young population as
well. If satellite cells have a limited proliferative capacity (~60
doublings), does a lifetime of intense exercise have a negative
influence on their ability to regenerate skeletal muscle as aging progresses?
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FUNCTIONAL RESPONSES TO DISEASE STATES |
Most myopathies have a molecular mutation that affects the
structural or cytoskeletal proteins in skeletal muscle. Duchenne muscular dystrophy (DMD) is the most common and the most devastating of
the muscular dystrophies (20, 55, 76, 82, 127). Disease progression and death are ultimately due to a failure of the myogenic satellite cells to maintain muscle regeneration (36, 80). DMD is a recessive X-linked disease that results in a null mutation at
the dystrophin locus (20, 82, 127). The absence of this cytoskeletal protein renders the muscle fiber extremely fragile. In
response to mechanical stress associated with repeated contraction, there is widespread degeneration. The satellite cells respond to the
injury by repopulating the injured skeletal muscle with defective
myofibers lacking dystrophin. This process results in continuous
degeneration-regeneration cycles and ultimately exhausts the satellite
cell pool (36, 80).
Clinical symptoms are apparent by 4-5 yr of age in boys with DMD
(10, 20, 127). DMD patients (4-5 yr of age) have been shown to undergo more skeletal muscle regeneration than that measured in a total of six normal patients over 60 yr of age (46).
These results were confirmed by Renault et al. (151), who
demonstrated that the proliferative life span of satellite cells
derived from a 9-yr-old DMD patient was approximately one-third of an
age-matched control. Proliferative fatigue or senescence of the
satellite cell population and the milieu of the DMD skeletal muscle may collectively impair the proliferative or regenerative capacity of this
cell population. In the DMD patient, increased levels of IGF binding
proteins (IGFBP) are released by fibroblasts. The elevated IGFBP
sequesters IGF-I, limiting its bioavailability for satellite cells and
ultimately resulting in increased skeletal muscle fibrosis
(120). The evidence to date demonstrates the tremendous
strain and ultimate failure of the satellite cell population to
adequately compensate for the persistent degeneration-regeneration process that is occurring in the DMD skeletal muscle.
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GENETIC MOUSE MODELS OF MYOPATHY |
A number of gene knockout mouse models and experimental methods
are available for studies of muscle regeneration and satellite cell
biology. Although the mdx mouse, which lacks dystrophin, has
provided important insights into the pathophysiology of DMD, the
myopathy in these animals does not represent the myopathic process of
DMD in humans (75). The mdx mice have a normal
life span, a temporally restricted myofiber degeneration, and adapt to
muscle degeneration with an expansion of the satellite cell pool and
muscle hypertrophy, thereby avoiding the compromised muscle function
that afflicts humans who lack dystrophin (51, 69, 116,
179). In addition to the spontaneous dystrophin mutation in the
mdx mouse, myopathic mouse models include genetically
engineered mouse strains with knockouts of utrophin (49,
69), MyoD (119), or MNF (63) crossed
into the mdx background. Each of these double mutant mouse
models exhibit features that more closely resemble DMD in humans,
including a severe myopathy, an impaired regenerative capacity, and a
decreased life span.
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MUSCLE REGENERATION MODELS |
An alternative strategy that has been successfully used for the
study of satellite cell activation, proliferation, regeneration, and
self-renewal is to experimentally produce a controlled skeletal muscle
injury. Strategies including crush (101), freeze
(40), or chemically induced injury (42, 63)
have all been successfully used to study satellite cell biology.
Perhaps the most extensive and reproducible muscle injury is the
delivery of cardiotoxin (purified from the venom of the Naja
nigricollis snake) into the hindlimb skeletal muscle of the
mouse (53, 63, 134). The intramuscular injection of 100 µl of 10 µM cardiotoxin into the gastrocnemius muscle results in
80-90% muscle degeneration (Fig. 5). After cardiotoxin-induced injury,
satellite cells become activated within 6 h of injury (Garry,
unpublished observations). In response to locally released growth
factors from injured myotubes and macrophages, the satellite cells
proliferate extensively within 2-3 days of injury (63,
64). Approximately 5 days after injury, the satellite cells
withdraw from the cell cycle and either self-renew or form differentiated myotubes that contain a central nucleus (63, 64). With the use of this cardiotoxin-induced injury protocol, the architecture of the injured muscle is largely restored within 10 days after injury (Fig. 5). The complete profile of intrinsic and
extrinsic cues that regulate the satellite cell population during
muscle regeneration remains unclear. Therefore, the use of reproducible
experimental injuries such as cardiotoxin-induced injury will be
important to evaluate and define the regulation of the satellite cell
population in molecular and physiological myopathic models. Additional
myonecrotic agents such as notexin have been used with similar success
(107), resulting in a well-characterized regenerative
response in skeletal muscle.
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Fig. 5.
Skeletal muscle response to toxin-induced injury. A:
transverse section of the adult gastrocnemius muscle stained with
hematoxylin and eosin at defined intervals following
cardiotoxin-induced injury. After cardiotoxin delivery, there is
evidence of extensive myonecrosis and edema of the myofibers (12 h;
denoted by a). A hypercellular response (proliferating
satellite cells and inflammatory cells) is observed within 2 days of
injury. Muscle regeneration is evident within 5 days of injury. At this
time, newly regenerated myofibers are evident as small, basophilic,
central-nucleated myofibers (denoted by b). The architecture
of the muscle is largely restored within ~10 days following injury.
The newly regenerated myofiber displays numerous centrally aligned
nuclei, demonstrating the fusion of many satellite cells to form a
single myofiber (hyperplasia) as denoted by the myofiber designated
c. B: schematic diagram emphasizing the temporal
pattern of satellite cell proliferation and muscle differentiation
following a chemically induced injury of adult mouse skeletal
muscle. Bar = 100 µm.
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SATELLITE CELLS AS MUSCLE PRECURSOR CELLS OR STEM CELLS |
TOP
ABSTRACT
INTRODUCTION
