Mature skeletal muscle is known to produce a host of growth factors such as insulinlike growth factor-1 (IGF-1) and hepatocyte growth factor (HGF) following a physiological stimulus such as overload or myotrauma following injury. IGF-1 has been shown to activate protein synthetic machinery by its interaction with the mammalian target of rapamycin (mTOR) (Frost and Lang 2011) and is a potent mitogen believed to be involved in cell recruitment to aid in tissue repair (Mourkioti and Rosenthal 2005). The expression of the MRFs appears to be regulated, in part, by the activity of locally produced isoforms of IGF-1 (Mourkioti and Rosenthal 2005; Chakravarthy et al. 2000). IGF-1 is a growth factor found in the circulation, produced in the liver and locally by the muscle fiber itself (Mourkioti and Rosenthal 2005). Three specific isoforms of IGF-1, IGF-1Ea, IGF-1Eb, and IGF-1Ec, have been described in muscle, and each may contribute to some extent to muscle regeneration/repair (Chakravarthy et al. 2000; Rotwein et al. 1986; Yang et al. 1996). IGF-1Ea transcribes the full-length IGF-1 protein, which is identical to that produced by the liver (Yang et al. 1996). IGF-1Eb may be involved in hypertrophy or muscle regeneration; however, the physiological role of IGF-1Eb in human skeletal muscle is currently unknown. IGF-1Ec or mechano growth factor (MGF) is a splice variant of IGF-1Ea that may have both autocrine and paracrine functions, stimulating satellite cell proliferation and muscle hypertrophy following muscle stretch or muscle damage (Yang et al. 1996; Hill and Goldspink 2003).

IGF-1 has been shown to activate cultured porcine satellite cells through an mTOR-dependent mechanism (Han et al. 2008) and enhance the proliferative potential of murine satellite cells by activating the phosphatidylinositol 3′-kinase/Akt signaling pathway (Galvin et al. 2003). In response to exercise or injury, IGF-1 splice variants increase within 24 h after injury in rats (Hill and Goldspink 2003; Hill et al. 2003); however, results from human studies are conflicting.

In humans, IGF-1Ec expression following isometric knee exercise can increase in as little as 2.5 h (Greig et al. 2006), suggesting that IGF-1Ec may be important for satellite cell proliferation. Information regarding the response of IGF-1Ea to exercise is equivocal, with some animal and human work demonstrating significant increases in IGF-1Ea expression as early as 24 h post-exercise or muscle damage (Hill and Goldspink 2003; Hill et al. 2003; Greig et al. 2006; Hameed et al. 2003), while others show a marked decrease in IGF-1Ea and total IGF-1 from 4 to 12 h post-exercise that remained unchanged after 96 h (Psilander et al. 2003; Bickel et al. 2005). In humans, the IGF-1 splice variants at the mRNA level (McKay et al. 2008) and the whole muscle protein level (Philippou et al. 2009), co-localized with Pax7 (using immunofluorescent techniques) (McKay et al. 2008), appear temporally related to MRF expression. IGF-1Ec is temporally co-expressed with Myf5, while IGF-1Ea and Eb are temporally co-expressed with MRF4, suggesting that alternate splicing of IGF-1 is associated with different phases of satellite cell progression (McKay et al. 2008; Philippou et al. 2009). Therefore, based on human and animal studies, it appears that IGF-1 may be a critical regulator of the myogenic response.

Other growth factors have been shown to influence the proliferative phase or differentiation of satellite cells in animals or in cell culture. Figure 10.2. shows a schematic representation of some of the major molecular regulators of the myogenic program and illustrates the timing of the MRFs with respect to the progression of satellite cells from quiescence to differentiation. These extracellular signaling molecules include basic fibroblast growth factor (bFGF), which has been demonstrated to induce activation/proliferation and inhibit differentiation (DiMario et al. 1989); nitric oxide (NO), which has been implicated in satellite cell activation, acting upstream of HGF to mediate the release of HGF (Tatsumi et al. 2002); Delta-1, which increases proliferation (Conboy et al. 2003); and myostatin and TGF-β, which inhibit activation and differentiation and maintain quiescence (McCroskery et al. 2003; Allen and Boxhorn 1987). Recently, angiotensin II (ANGII) has been implicated as an activator of quiescent satellite cells in vivo and in culture (Johnston et al. 2010). The actions of ANGII appear to regulate satellite cells through the angiotensin II receptor sub-type 1 (AT1), and may also play an instrumental role in satellite cell migration following muscle injury (Johnston et al. 2010). Platelet-derived growth factor (PDGF) and endothelial growth factor (EGF) also appear to increase satellite cell proliferation in culture (Hawke and Garry 2001). The stark reality is that there are many factors that influence satellite cell regulation, many of which are likely yet to be discovered. This reality underlies the complexity of understanding satellite cell regulation and emphasizes the challenge of understanding age-related satellite cell dysfunction. A growing area of interest is the role that inflammation and inflammatory markers play in regulating satellite cell function. Not surprisingly, the role of inflammatory markers on satellite cell function is not well understood.

In young adults, myotrauma due to exercise or injury may induce damage to contractile proteins and myofibrils, or larger ultrastructural disruptions, which initiates a significant inflammatory response (Smith et al. 1994b; Beaton et al. 2002). Macrophage infiltration, which is responsible for the removal of cellular debris, is generally accompanied by lymphocytes, which coordinate the inflammatory response and importantly release factors that stimulate satellite cell proliferation (Allen et al. 1995; Cantini et al. 1995). Invading T lymphocytes can bind adhesion molecules on damaged muscle fibers and release a host of cytokines including leukemia inhibitory factor (LIF) and interleukin-6 (IL-6). In addition, damaged muscle fibers secrete several cytokines (i.e., IL-6) and growth factors (i.e., IGF-1) that may act as chemoattractants for inflammatory cells and satellite cells, augmenting the rapid repair response (Johnston et al. 2010; Pedersen and Febbraio 2008; Broholm et al. 2008; Gallucci et al. 1998; Tomiya et al. 2004). In humans, administration of nonsteroidal anti-inflammatory medication (NSAID) after damaging exercise inhibits the exercise-induced increase in satellite cells observed in controls (Mikkelsen et al. 2009; Mackey et al. 2007), illustrating that inflammation may be a key factor regulating the satellite cell response.

Cytokines released by inflammatory cells have been shown to act on satellite cells in vitro, stimulating proliferation (Cantini et al. 1995). Of the factors released, members of the interleukin-6 family of cytokines, IL-6 and LIF, appear to be instrumental in regulating satellite cell proliferation (Cantini et al. 1995; Spangenburg and Booth 2002; Austin et al. 1992). Both LIF and IL-6 bind the gp130 receptor, activating Janus-activated kinase (JAK) 2, which induces the phosphorylation of signal transducers and activators of transcription (STAT)-3 (Spangenburg and Booth 2002; Rawlings et al. 2004). Phosphorylated STAT3 (pSTAT3) forms a homodimer (or heterodimer with other STAT proteins), which translocates to the nucleus and induces the transcription of target genes such as Cyclin D1 and cMyc, which are instrumental in the initiation of the cell cycle (Rawlings et al. 2004). Importantly pSTAT3 also targets the IL-6 gene; thus, the release of IL-6 and LIF induces the production of IL-6 in target cells, creating an autocrine/paracrine loop influencing satellite cell proliferation (Rawlings et al. 2004). Until recently these findings had only been validated in cell culture models. In a landmark study by Serrano and colleagues (Serrano et al. 2008), IL-6 was shown to be an essential regulator of satellite cell function in vivo in a murine animal model. This study provided mechanistic evidence supporting the role of IL-6 as a regulator of satellite cell proliferation and that in the absence of IL-6 (IL-6 ^−/− mice), satellite cell proliferation and myonuclear accretion were severely blunted (Serrano et al. 2008). Furthermore, the deficiency in proliferation observed in the IL-6^−/− satellite cells could be rescued by the ectopic addition of IL-6 and IL-6-mediated proliferation via the IL-6/STAT3 axis (Serrano et al. 2008). This study provides strong evidence to support the role of inflammatory cytokines in regulating muscle stem cell activity.

Aging is associated with alterations in basal levels of circulating inflammatory mediators such as IL-6 and TNFα and a decrease in circulating growth factors. The changes associated with normal tissue aging confer a negative impact on the muscle stem cell compartment, which may impact the ability of the skeletal muscle to adapt and regenerate. The remainder of this chapter focuses on several aspects of the aging process in relation to the function and control of the muscle stem cells.

Aging is a complex process and is associated with many physical and metabolic alterations that lead to the progressive loss of function over time (Roubenoff 2003). Sarcopenia, which is an age-related condition that includes a progressive loss of muscle mass and strength, leads to the loss of functional capacity and an increase in morbidity (Roubenoff 1999; Beccafico et al. 2007). The progressive nature of age-related muscle wasting leads to an increased incidence of injury and loss of autonomy, resulting in a significant loss in quality of life and increased risk of all-cause mortality (Roubenoff and Hughes 2000; Melton et al. 2000). Moreover, the economic impact of sarcopenia is staggering. Common age-related injuries are often associated with falls due to a loss of strength and stability. Falls represent approximately 65 % of injuries, 85 % of injury-related hospital admissions, and 58 % of injury-related deaths in individuals over the age of 65. These statistics translate into health-care costs of approximately $1 billion dollars annually in Canada and as high as $26 billion dollars annually in the United States (Janssen et al. 2004). Similar trends have been observed in Australia, Great Britain, and Europe. Thus understanding the cellular mechanisms that lead to the onset and progression of sarcopenia is necessary to propose effective countermeasures.

The normal aging process has a profound effect on the structure and function of the skeletal muscle. Aged muscle undergoes progressive and seemingly unavoidable atrophy, with a decreased cross-sectional area accompanied by a decrease in muscle fiber number and fiber size (Grounds 1998; Koopman and van Loon 2009). Importantly, muscle function or quality appears to be reduced in aged muscle, displaying a decrease in power and maximum force production even when corrected to cross-sectional area (Brooks and Faulkner 1988). In addition, aged muscle is far more susceptible to macro and micro damage compared to young muscle, and, central to this chapter, aged muscle displays a decreased capacity for growth and regeneration (Grounds 1998; Brooks and Faulkner 1996). Satellite cells have been implicated as a significant factor contributing to the inability of the muscle to repair/remodel. An age-related impairment of the myogenic program (either impaired activation of or progression through the myogenic program) is hypothesized to be a significant contributor to satellite cell dysfunction, leading to the onset or progression of sarcopenia. In adult muscle, satellite cells comprise only a small fraction of the total myonuclei (~2 %) yet possess an incredible proliferative capacity, capable of repairing extensive muscle injury. The size of the satellite cell pool is thought to remain relatively constant throughout adult life; however, it has been suggested that in the later stages of life, the size of the satellite cell pool begins to decline, impairing the regenerative capacity of muscle. Although it is unclear at present whether there is an age-related reduction in the satellite cell pool size, it is clear that aging is accompanied by an impairment in the response of satellite cells to myotrauma in humans (Dreyer et al. 2006; McKay et al. 2013; McKay et al. 2012) and animals (Conboy et al. 2003). Several studies have attributed both intrinsic factors of the satellite cells (Jejurikar et al. 2006) as well as extrinsic factors such as age-related alterations to the satellite cell niche (Conboy et al. 2003; Carlson and Conboy 2007) as major influences on the dysfunction of the satellite cell population with aging.

There is a progressive decline in the satellite cell pool after birth from ~30–50 % of total myonuclei to a relatively stable ~2–4 % of total myonuclei in adolescences and into adulthood. Whether or not aging is associated with a reduction in the total number of satellite cells has been the focus of many studies in animals and in humans and would be the simplest explanation as to why older adults suffer significant skeletal muscle loss. Although there is significant controversy in this area, there are several key studies in animals that highlight an age-related decline in the satellite cell pool. Shefer et al. (Shefer et al. 2006) compared the satellite cell pool in four groups of mice aged 3–6 months (young), 7–13 months (adult), 19–25 months (old), and 29–33 months (senile). Their data demonstrate a 60 % decline in satellite cells in the extensor digitorum longus (EDL) muscle between young and old mice with a further 17 % reduction between the old and senile groups (Shefer et al. 2006). Similar results were observed in the soleus muscle, and it was concluded that the satellite cell pool in the young was significantly larger than that of all the three other groups. Furthermore, when grown in culture, a more robust growth rate was observed in satellite cells obtained from young muscle as compared to senile muscle. Other studies in support of these findings have also reported decreased growth rates as well as increased susceptibility for satellite cells to undergo apoptosis in vitro and in vivo compared to the young (Jejurikar et al. 2006; Shefer et al. 2006; Day et al. 2010). Single muscle fiber analysis revealed that satellite cells, expressing the marker Pax7, were markedly reduced in aged muscle and had a reduced myogenic capacity in culture (Collins et al. 2007). In fact, approximately 10 % of aged myofibers did not possess any Pax7-positive cells (satellite cells). Importantly, the aged satellite cells demonstrated a marked inability to renew the progenitor pool undoubtedly contributing to the observed reduction in satellite cell number. A rather interesting observation by Collins et al. (2007) described a subpopulation of satellite cells that appeared unaffected by the aging process. Although the majority of aged satellite cells displayed altered myogenic properties, a population of relatively rare muscle stem cells was able to produce large colonies of functional satellite cells, which retained the ability to self-renew in vitro (Collins et al. 2007). This population likely represents a very small proportion of the satellite cell pool and may be a more primitive myogenic progenitor cell, which, for reasons unknown, has been able to avoid the adverse effects of aging. It may be that these cells represent a more primitive myogenic progenitor subpopulation, which retains a similar level of potency as satellite cells from young muscle. There is significant heterogeneity in the satellite cell population (Biressi and Rando 2010), and it is possible that, much like in the bone marrow, there is a small group of stem cells that rarely divide and whose progeny is mainly responsible for amplifying in response to stimuli and self-renewing to maintain the progenitor pool. It is of note, however, that the notion that aging is associated with a loss in the size of the satellite cell pool does not go unchallenged. Several studies do not report a decline in total satellite cell number in the muscle of aged mice (Conboy et al. 2003; Dreyer et al. 2006). This may be a result of different enumeration methods or molecular markers used to quantify the satellite cell pool or species of animal used. In humans, however, it has been unequivocally shown that aged muscle (>65 years) has a reduced satellite cell pool. Recently Verdijk et al. (Verdijk et al. 2007; Verdijk et al. 2009), using the cell marker N-CAM (neural cell adhesion molecule), demonstrated that the satellite cell pool in older adults was significantly lower compared to young men, and this was mainly a consequence of the loss of satellite cells associated with type II myofibers. This was subsequently confirmed using both Pax7 immunofluorescent analysis and flow cytometry in older adults (~70 years) (McKay et al. 2012, 2013). Collectively these studies demonstrate an approximate 35 % reduction in the satellite cell pool in 70-year-olds compared to 25-year-olds, the bulk of which is made up of a marked reduction in type II-associated satellite cells (McKay et al. 2012, 2013; Verdijk et al. 2007, 2009, 2012). These studies report an approximate 30 % reduction in muscle strength, 25 % reduction of type II muscle fiber cross-sectional area (CSA), and a 20 % lower type II fiber myonuclear domain compared to young lean body mass-matched controls. Importantly these studies provide evidence that the reduced type II fiber-associated SC content in the elderly may be an important factor in the etiology of type II fiber atrophy and progression of sarcopenia (Verdijk et al. 2007). Whether the absolute number of satellite cells is an important factor for muscle aging remains a somewhat contentious issue; however, the literature clearly demonstrates that satellite cells in aged muscle become dysfunctional as a consequence of the aging process. The dysfunction can be classified as intrinsic to the stem cells themselves or extrinsic, relating to factors in the stem cell niche and circulating factors known to regulate satellite cells. It appears that the combination of these extrinsic and intrinsic factors is important to the overall function or dysfunction of the muscle stem cell population.

Muscle stem cells remain in a quiescent state on the periphery of the myofiber and become activated when sufficient cellular cues from the microenvironment initiate entry into the cell cycle. A major factor influencing the success of this process is the ability of the cell to recognize these signals and activate the appropriate cell signaling pathways. Several studies have identified key factors that appear to be altered by the aging process. Furthermore, nuclei in newly fused myoblasts must function properly to allow for adequate tissue healing and to respond appropriately to physiological stimuli for the induction of adaptation. In aged muscle it appears that all of these aspects may be impaired to some degree. Several theories have been proposed to explain how quiescent cells outside the sarcolemma could acquire age-related damage. The most prominent of these theories involves oxidative damage to DNA and cellular components from years of exposure to reactive oxygen species (ROS) (Beccafico et al. 2007; Cefalu 2011; Mecocci et al. 1999). Satellite cells and myotubes (fused differentiated myoblasts) from aged humans display an age-dependent increase in lipid peroxidation and impaired calcium handling in in vitro experiments suggesting that acquired damage throughout life impairs the basic cellular processes in these cells which may contribute to the poor adaptive response of aged muscle (Beccafico et al. 2007).

The satellite cell niche and the systemic milieu have been identified as factors in age-related satellite cell dysfunction. The satellite cell niche or microenvironment that supports the satellite cell helps regulate cell activity through physical contact and direct interaction with satellite cell membrane proteins (Kuang et al. 2008; Gopinath and Rando 2008). The basal lamina is one of the main niche components, which is in constant interaction with extracellular space and circulation. It is mainly composed of type IV collagen and several structural proteins, proteoglycans, and glycoproteins such as fibronectin and laminin (Woodley et al. 1983).