Although endurance training is associated with high-repetition–low-resistance exercise, significant muscle damage can occur if the duration or mode of exercise is extreme. For example, both marathon running and downhill running (eccentric exercise) can lead to significant muscle fiber damage, and many studies use eccentric exercise as a modality to induce muscle injury.
In contrast to endurance training, resistance exercise training is associated with high-intensity–low-repetition workloads leading to increases in muscular strength, power, and oxidative capacity, with little change in aerobic capacity. The workloads placed on skeletal muscle during resistance training are at or near maximal capacity, and as such produce significant perturbations to the skeletal muscle fibers and the associated extracellular matrix.
The muscle damage associated with intense exercise (regardless of exercise type) includes disruption to the extracellular matrix, basal lamina, and sarcolemma, resulting in the release of intracellular proteins such as myoglobin and creatine kinase. Within the muscle fiber, damage to contractile and cytoskeletal proteins can also occur, leading to decreased fiber tension and even death of the muscle fiber. Interestingly, whereas both endurance and resistance exercise can result in muscle injury, resistance training is more likely to be associated with increases in fiber cross-sectional area. The reasons for this point to differences in the integration of hormonal, metabolic, mechanical, neuronal, and immune responses, which are all likely involved in the distinct transcriptional responses that characterize endurance and resistance training.
RESIDENT MUSCLE STEM CELL POPULATIONS
Adult skeletal muscle contains identifiable cell populations with stem cell–like characteristics. One of these cell populations, the myogenic progenitor cells (MPCs), are also known as satellite cells based on their location at the periphery of adult myofibers (3). These undifferentiated progenitor cells are the most thoroughly characterized of the resident muscle stem cell populations. The MPCs are quiescent in the unstressed muscle, but can reenter the cell cycle (become “activated”) in response to signals associated with muscle damage. After activation, these cells will proliferate and migrate to the site of injury to repair or replace damaged myofibers by fusing together and/or fusing to existing myofibers (Fig. 1A) (6).
The fusion of MPCs to existing myofibers is critical for large increases in myofiber cross-sectional area, and works on the premise of the myonuclear domain theory. This theory suggests that the myonucleus controls the production of mRNA and proteins for a finite volume of cytoplasm, such that increases in fiber size (hypertrophy) must be associated with a proportional increase in myonuclei, which are contributed from the MPC population (Fig. 1B). Importantly, the MPCs are self-renewing, such that a residual pool of these cells is reestablished after each discrete episode of muscle injury, and therefore capable of supporting additional rounds of regeneration. Whereas the MPC displays some similarities to other adult stem cell populations (such as self-renewal and a limited capacity to adopt alternative lineages), it is largely assumed that these cells are “committed” to the skeletal muscle lineage.
An additional population of cells with stem cell–like characteristics has recently been identified within numerous adult tissues, including skeletal muscle. These muscle stem cells can be isolated using dual-wavelength flow cytometric analysis (FACS) based on their ability to efflux the DNA dye, Hoechst 33342. Muscle stem cells isolated using FACS analyses are termed SP cells because they appear as a “side population” on the FACS profile (Fig. 2A). Muscle SP cells are far rarer than the MPCs within resting adult skeletal muscle (<0.2 vs 2–5% of all muscle nuclei), and have been shown to display a greater ability to adopt other cell lineages (plasticity) than the MPC population (7,11).
A number of studies have recently identified subpopulations within the muscle SP cell populations that display differential capacities for self-renewal, plasticity, proliferation, and differentiation (4). Whether these subpopulations represent distinct cell populations, or whether they are the same cell population adapting to changes in their microenvironment, is still under investigation.
ARE MUSCLE SP CELLS INVOLVED IN SKELETAL MUSCLE REGENERATION?
Virtually all mammalian tissues have been demonstrated to contain a SP cell complement. A complete discussion of the contribution and potential role of these nonmuscle SP cells in skeletal muscle regeneration is beyond the scope of this review; the reader is referred to Charge and Rudnicki (2) and Grounds et al. (4) for a review of the potential contribution of these other stem cell populations in muscle repair.
Skeletal muscle hypertrophy and regeneration were previously believed to be mediated solely through the MPCs. There is increasing evidence, however, to suggest that, given some circumstances, muscle (and even nonmuscle) SP cell populations can contribute to the regeneration of injured skeletal muscle (Fig. 3). For example, Gussoni et al. (5) injected muscle SP cells into the tail vein of lethally irradiated myopathic (mdx) mice and demonstrated the ability of a small number of these cells (<0.5%) to contribute to regenerated muscle, and also found evidence to suggest that transplanted muscle SP cells may give rise to quiescent MPCs. Consistent with these findings, an intriguing study by LaBarge and Blau (9) found that bone marrow–derived SP cells could contribute to skeletal muscle repair in lethally irradiated mice. Furthermore, these transplanted cells gave rise to quiescent MPCs that were capable of contributing to the formation of approximately 3.5% of the regenerated myofibers in response to subsequent exercise-induced damage.
Anecdotal support for the role of muscle SP cells in skeletal muscle repair comes from the finding that muscle SP cell number increases significantly in response to muscle injury (Fig. 2). Furthermore, in a mouse model with impaired muscle regenerative capacity (Foxk1 mutant mice), there is a reduction in their muscle SP cell number, and the injury-induced increase in the SP cell number is significantly blunted (Fig. 2) (11). Additionally, Rehman et al. (12) demonstrated an increase in circulating endothelial progenitor cells with a single bout of exhaustive exercise, leading the authors to speculate that these cells were mobilized for the purpose of repair and angiogenesis in response to intense exercise. This finding is in agreement with the increase in muscle SP cell number with muscle injury, which may be mobilized for the purpose of contributing to skeletal muscle regeneration (11).
Taken together, these studies provide some support for the hypothesis that the SP cell population may be precursors to the MPCs and, given the appropriate extracellular milieu and cell–cell interactions, that SP cells are capable of adopting a myogenic lineage and contributing to skeletal muscle regeneration. However, it has yet to be elucidated whether endogenous SP cells, particularly muscle SP cells, are involved in the hypertrophy or exercise-induced regeneration of normal skeletal muscle. To date, the contribution of endogenous SP cells to these processes appears minimal, although from a therapeutic perspective, lethal irradiation followed by significant muscle injury, or increasing local growth factor levels, may increase their overall contribution (4).
EXTRINSIC CUES REGULATING MUSCLE STEM CELLS DURING REGENERATION AND HYPERTROPHY
The ability of skeletal muscle to respond to stressors such as strenuous exercise is mediated by a complex array of extrinsic and intrinsic cues. A critical step in this adaptability involves modulating the timing, availability, and receptor density for the muscle growth factors. Although there are obvious physiological, cellular, and molecular distinctions between skeletal muscle regeneration and hypertrophy, both processes share similarities regarding MPC activation, proliferation, and differentiation. With skeletal muscle hypertrophy, the MPCs will fuse to the existing myofibers, essentially donating their nuclei, whereas regeneration from more extensive muscle damage may result in the fusing together of MPCs to generate a new myofiber. Newly generated myofibers are identified by their centrally located nuclei (Figs. 1 and 4).
Because the role and regulation of the muscle SP cell population are still largely unknown, this section focuses on the MPC and its role in the adaptability of skeletal muscle to exercise.
The activation of MPCs is characterized by alterations in their morphology (increased cytoplasmic to nuclear ratio, increased cytoplasmic organelles, and reduced heterochromatin) and their adhesion characteristics to the mature myofiber. A potential mechanical link between myofiber damage and MPC activation has recently been uncovered (14). In this model, myofiber damage leads to the bolus release of nitric oxide, which mediates the release of active hepatocyte growth factor from its heparin sulfate chains on the extracellular matrix and surrounding myofibers. The release of hepatocyte growth factor in response to muscle injury occurs rapidly (order of minutes), and is proportional to the degree of muscle injury. Hepatocyte growth factor binding to its receptor, c-met, located on the MPC plasma membrane, is one of the earliest events in MPC activation, and leads to a cascade of signaling events promoting cell proliferation and changes in focal adhesion (Fig. 5).
Recently, an isoform of insulin-like growth factor, termed mechanogrowth factor, has been identified; it is released early (minutes to hours) after increased loading or stretch of the muscle (1). The role played by mechanogrowth factor in MPC activation is currently being investigated, but based on its early release, it may be involved in promoting transcriptional changes associated with preparing the cell for increased cellular proliferation.
Proliferation and Differentiation
There are numerous studies demonstrating the potential involvement of various growth factors in regulating the MPCs in vitro (6). Although these in vitro studies do provide insight into the regulation of the MPC, the effects observed are not always consistent with in vivo studies. These differences may be attributed to variations in growth factor timing, availability, and receptor density.
The degree of immune response associated with strenuous exercise is proportional to the degree of mechanical damage to the muscle; endurance exercise is associated with a more systemic immune response, whereas resistance exercise is associated with a more localized immune response. Within a few hours after skeletal muscle damage, circulating neutrophils are increased and the damaged tissue attracts macrophages, which respond by releasing chemokines and cytokines that will act as an attractant for the migration of neutrophils, monocytes, and MPCs, as well as increase blood vessel permeability, allowing for an increase in fluid and protein transition into the extracellular space. Among the cytokines associated with the inflammatory response, leukemia inhibitory factor has been demonstrated to increase significantly in response to exercise-induced injury, and is capable of increasing MPC proliferation in vitro. Interestingly, the exogenous administration of leukemia inhibitory factor to skeletal muscle can promote MPC proliferation, myofiber hypertrophy, and improved regeneration after injury. Collectively, the inflammatory cytokines, and in particular the IL-6 family of cytokines (of which leukemia inhibitory factor is a member), appear to play an integral role in the repair process after myotrauma (15).
Of all the muscle growth factors, insulin-like growth factor-I (IGF-I) has been the most thoroughly characterized during muscle hypertrophy. In response to resistance training, IGF-I secretion from skeletal muscle is elevated, and promotes proliferation and fusion of the MPCs. The ability for IGF-I to mediate both MPC proliferation and differentiation may be caused by the capacity to signal through two distinct signaling cascades. The proliferative effects of IGF-I have been largely attributed to the Ras/Raf/MEK/ERK pathway, whereas the PI3K/AKT signaling pathway has been proposed to mediate the differentiation cues (10). Although cross-talk between these two cascades is likely, recent evidence has demonstrated a key role of the PI3K/AKT pathway in mediating MPC proliferation (10). The essential role played by IGF-I in MPC regulation has been demonstrated in vivo, where superfusion or overexpression of IGF-I results in skeletal muscle hypertrophy and prevention of age-related sarcopenia 1.
In general, the transforming growth factor (TGF)-β family (including myostatin) of cytokines is proposed to inhibit MPC proliferation and differentiation, primarily through silencing the transcriptional activation of the MyoD family members. In response to muscle damage, circulating and local TGF-β levels are elevated. The inhibition of MPC proliferation and differentiation by TGF-β in response to muscle damage may seem counterintuitive. Sakuma et al. (13) suggest that during regeneration from muscle injury, TGF-βII ligand and receptor levels are reciprocally expressed, and that this reciprocal expression pattern may result in the initial promotion of cellular proliferation followed by enhanced muscle differentiation. In addition to modulating MPC activity, they are involved in promoting normal skeletal muscle architecture by regulating local collagen synthesis in tendon-related connective tissue (13).
CHANGES IN EXTRINSIC CUES WITH CHRONIC TRAINING
As stated previously, both endurance and resistance exercise result in varying degrees of muscle fiber damage. In response to the chronic perturbations placed on the skeletal muscles during resistance training, there are significant increases in fiber size, myonuclei, and MPC number (8). These alterations are not observed with chronic endurance exercise training, although significant myofiber turnover can still occur (3). This dichotomy could be explained, in part, by the observation that elevations in plasma testosterone and growth hormone levels appear to be more dependent on exercise intensity than exercise volume. Increases in these hormones may mediate increases in IGF-I, resulting in the elevated MPC number and myofiber hypertrophy observed with chronic resistance training or exogenous testosterone administration.
Despite the tremendous interest in hormones, growth factors, and exercise, there is limited information on changes in these factors in response to chronic training. Furthermore, although changes in circulating hormone levels may occur in response to either resistance or endurance training, it is also important to identify whether reciprocal changes in receptor density occurs. Also of interest is the role and regulation of the extracellular matrix with chronic training. For example, does the extracellular matrix become more or less sensitive to muscle damage with training?
The adaptability of skeletal muscle in response to a range of stressors is remarkable. Although we have learned much about the general physiology of this organ system, our understanding of the mechanisms regulating this adaptability remains a fertile area of research.
The author recognizes the research efforts of those included and omitted (only because of space considerations) who have contributed to this field of study. In some cases, review articles have been used to direct the reader to original articles or more comprehensive summaries of the literature. A special thanks to Drs. R. Allen, F. Booth, S. Kanatous, and A. Meeson for helpful discussions and critical reviews of this manuscript. The author’s work is supported by grants from the Canadian Foundation for Innovation, the Ontario Innovations Trust, and the Hospital for Sick Kids Foundation.
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