Sci. Aging Knowl. Environ., 3 December 2003
Vol. 2003, Issue 48, p. pe34
[DOI: 10.1126/sageke.2003.48.pe34]

PERSPECTIVES

Does the Road to Muscle Rejuvenation Go Through Notch?

Jeffrey Boone Miller, and Charles P. Emerson Jr.

The authors are at the Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472, USA. E-mail: emersonc{at}bbri.org (C.P.E.)

http://sageke.sciencemag.org/cgi/content/full/2003/48/pe34

Key Words: sarcopenia • muscle repair • Notch • Delta • skeletal muscle • regeneration

Therapies that could ameliorate or reverse the loss of skeletal muscle during aging would be a boon to the elderly population. In humans, as much as one-third of muscle strength and mass may be lost between the ages of 30 and 80 years. This aging-associated loss, termed sarcopenia (see Hepple Perspective), leads to impaired mobility and frailty in a significant fraction of elderly people (1, 2). Some of the loss of muscle mass with aging may result from a decrease in the ability of injured muscle to repair itself. Skeletal muscles often incur injuries during normal use and when overworked. These injuries are repaired by muscle precursor cells (satellite cells) that reside in a quiescent state in uninjured skeletal muscles. After muscle injury, satellite cells proliferate and either fuse to and repair injured muscle fibers or produce entirely new muscle fibers (Fig. 1). However, this regeneration after injury is markedly impaired in aged muscles (3, 4).



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Fig. 1. Cell biology of muscle repair. In uninjured muscle, the muscle precursor cells, called satellite cells, are not actively dividing, and the myonuclei in the multinucleate muscle fibers are peripherally located, near the plasma membrane. Shortly after injury, satellite cells begin to proliferate. Within a few days, the satellite cells either fuse to and repair injured muscle fibers that have survived the injury or fuse with each other to form entirely new muscle fibers that replace dead fibers. As repair nears completion, the satellite cells again become quiescent, and the regenerated fibers, although they reach normal size and are innervated, have myonuclei that are located in the center of the fibers, rather than on the periphery. Photographs of hematoxylin and eosin-stained, transverse cross-sections of a mouse tibialis anterior muscle are shown in the three lower panels, with diagrams of the corresponding cellular events shown in the upper three panels.

 
In the 28 November 2003 issue of Science, Conboy et al. (5) report findings that significantly advance our understanding of how aging affects skeletal muscle. These investigators describe experiments that identify the Notch/Delta signaling system (Fig. 2) as an important regulator of muscle regeneration. In particular, they show that Notch signaling is impaired in aged muscle as a result of a lack of up-regulation of the Delta ligand upon injury. Furthermore, they show that targeted alterations of the Notch/Delta system can markedly improve the regenerative capacity of aged muscle to near that seen in young muscle.



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Fig. 2. Signaling in the Notch/Delta system. When Delta displayed on one cell interacts with Notch on an adjoining cell, a protease cascade is activated. As a result, ADAM17 (a Disintegrin and Metalloproteinase number 17) cleaves Notch into the Delta-binding fragment (blue) and the Notch extracellular truncation (NEXT) fragment. The Delta-binding fragment is endocytosed with Delta into the Delta-expressing cell. Cleavage of NEXT by {gamma}-secretase releases the NICD to participate in a transcription factor complex. [Figure reprinted by permission from (28)]

 
Signaling through the families of Notch receptors and Delta and Jagged ligands is important in many tissues during both development and adult life (Fig. 2) (6, 7). Humans have at least four Notch receptors (Notch-1 to -4) and at least five ligands (Jagged-1 and -2 and Delta-1, -3, and -4), plus the possibility of additional ligands, such as F3/contactin, which functions as a Notch ligand in oligodendrocyte differentiation (8). Upon ligand binding, an intracellular domain of Notch (NICD) is released, translocates to the nucleus, and forms a transcriptional regulatory factor by complexing with additional proteins such as the CSL (CBP1, Suppressor of Hairless, Lag-1) polypeptides. Both the ligand and the receptor are cell surface proteins, so that Notch signaling depends on intercellular interactions (Fig. 2). Among the genes in which transcription is mediated by Notch are members of the Hairy/Enhancer of split family of transcription factors. Altered expression of members of the Notch/Delta families underlies several human diseases, including T cell leukemia and vascular abnormalities (9).

In skeletal muscle, the Notch pathway is known to play roles in both embryonic myogenesis and adult muscle. In the vertebrate embryo, Notch/Delta signaling is required for proper terminal differentiation of somitic myocytes, which are the first skeletal muscle cells to form during development, and of muscle cells in the limb buds (10, 11). In additional studies with cultured cells, Notch signaling was identified as a regulator of the proliferation and differentiation of muscle precursor cells obtained from postnatal muscles (12).

In their new work (5), Rando's group has extended the analyses of Notch function to aged muscle by identifying Notch signaling defects that underlie the much poorer repair of injured muscle in aged mice (23 to 24 months old) as compared with mature (5 to 7 months) or young (2 to 3 months) mice. Small muscle injuries were induced by localized freezing or by needle insertions, and the success of regeneration was examined by quantitative histology 1 to 5 days after injury. In addition, the number of cells that proliferated during the regeneration episode was determined by immunohistological localization of cells that incorporated bromodeoxyuridine.

As one part of their study, Conboy et al. (5) found that the satellite cells from aged muscle were as numerous and as able to form myotubes as satellite cells from younger muscles, although these myoblasts from older muscles proliferated much less. These results, which are consistent with some previous studies of other aged muscles [reviewed in (3, 4)], identify the loss of proliferation capacity of satellite cells as a key cellular mechanism underlying the poorer regeneration capacity of aged muscles.

Because proliferation defects appeared to underlie decreased repair in aged muscle, the first key finding of Conboy et al. (5) was that Delta, which is a regulator of muscle cell proliferation, is expressed after injury at much lower levels in aged muscles than in younger muscles. The investigators found that young muscles showed a large up-regulation of Delta upon injury, with Delta appearing on the surface of satellite cells and multinucleate muscle fibers, as well as on interstitial cells that were not further identified. Delta was up-regulated not only at the immediate injury site but also in a moderately sized area around the injury, which is consistent with the idea that injured muscle fibers release a soluble factor that induces Delta up-regulation in responsive satellite cells and muscle fibers. In contrast to the robust up-regulation of Delta in injured young muscles, satellite cells and muscle fibers in injured aged muscles failed to significantly up-regulate Delta expression after injury. Because Delta is a regulator of satellite cell proliferation, these findings suggest that targeted alteration of Notch and Delta would modify muscle repair.

This possibility was directly confirmed by two additional sets of experiments. First, when Notch signaling was inhibited by local administration of a Jagged-Fc fusion protein, the repair of young muscle was significantly inhibited, so that it resembled the poor repair of aged muscle. Second, when Notch signaling was increased by local administration of a specific monoclonal antibody that activates Notch, the repair of aged muscle was markedly improved, so that it approximated the more successful repair in young muscle. This result plus the direct measurements of Notch concentration by the authors showed that Notch concentration is normal in aged muscle, but Notch activation is impaired because of a lack of ligand. Thus, the authors concluded that "inadequate activation of Notch-1 by Delta contributes to the loss of regenerative potential in old skeletal muscle." They speculate that a similar loss of regenerative capacity upon aging by other, nonmuscle tissues may similarly be controlled by Notch signaling.

These encouraging results suggest that the road to muscle rejuvenation may indeed go through Notch; but before we reach a final answer, several questions raised by the new results will need to be investigated.

One open question is whether regenerated myotubes in aged muscle are functional and stable. The initial analyses of Conboy et al. (5) were limited to the first 5 days after injury. In humans, untreated, injured, aged muscles are known to be unable to restore normal histology and function even after months-long periods of regeneration. It will be important for the development of therapeutics that the regenerated myotubes in Notch-activated aged muscles be stable over the long term, properly innervated, and functionally integrated into the whole muscle. The issue of innervation is important, because motor neurons are lost during aging, so that in some cases a lack of motor neurons for proper innervation may inhibit the functional regeneration of aged muscles [reviewed in (3, 4)]. Lack of innervation might be particularly problematic in injuries larger than those studied by Conboy et al. and in the attempt to halt or reverse sarcopenia.

Although Notch signaling can improve the repair of injury in the aged muscle model of Conboy et al., it remains to be determined whether the loss of muscle mass and strength that occurs during normal aging, or possibly in diseased muscles, could be improved by long-term Notch activation. Some neuromuscular diseases, including Duchenne muscular dystrophy, are characterized by cycles of muscle degeneration and regeneration, and so it is possible that Notch activation could ameliorate some aspects of the myopathology. This possibility could be tested by determining how activators and inhibitors of Notch alter the course of disease and tissue damage in mouse models of the human dystrophies. Furthermore, the proliferation capacity of satellite cells is much lower in aged and dystrophic muscle than in normal muscle (13, 14). The muscle wasting seen in disease-related cachexia would be an additional target for testing the effects of Notch activation. If, in addition to accelerating the rate of proliferation, Notch activation could increase the number of divisions that satellite cells from aged or diseased muscles are capable of, then Notch activation might be more likely to be a useful ameliorative therapy. This possibility could be tested by determining the maximum number of progeny obtained from cloned muscle precursor (satellite) cells with and without Notch activation (13, 14).

The cell-type specificity of Notch activation also remains to be determined. Which cells in injured muscle are responding to Notch activation? Because satellite cells show up-regulation of Delta in injured muscle and respond to Notch activation, they are likely to be the most important target. However, also consistent with the data of Conboy et al., is the idea that cells in vascular and connective tissues, as well as injured muscle fibers and even nearby uninjured muscle fibers, also respond to Notch activation. Although perhaps less important in the small injuries (needle tracks) and relatively short repair times studied by Conboy et al., proper formation of capillaries and inhibition of scarring are requirements for successful muscle regeneration after large injuries [reviewed in (3, 4)].

Downstream components of the Notch pathway need to be delineated and any muscle cell-specific components identified, because all such muscle cell-specific entities would be potential targets for the development of therapeutic agents. In addition, some endogenous ligands that normally activate Notch in injured muscle may have yet to be identified. Which Deltas and Jaggeds are expressed in muscle? Which of them activate Notch upon injury? Are there other ligands that are muscle-specific? Also important is identification of the mechanisms by which the Notch pathway, particularly loss of the Delta ligand, is adversely affected by aging. Is the loss of Delta the only change in the Notch pathway with aging? If so, what is the mechanism underlying such a specific loss of function; and if not, what other regulators of Notch signaling are lost and by what mechanisms? Numerous cellular functions are known to be compromised in aging tissue (such as loss of mitochondrial function, loss of redox balance, and accumulation of modified proteins), and so it should be possible to generate testable hypotheses about how aged muscle loses the ability to up-regulate Delta in response to injury.

Several studies over the past few years have identified treatments distinct from Notch activation that also may prove successful in ameliorating sarcopenia or improving muscle regeneration after injury. For example, long-term clinical trials have shown that treatment with human growth hormone can improve muscle function in the elderly, although with substantial side effects (see "Strong Muscles, Strong Tumors?" and "Lean Yes--But Mean?") (15, 16). In addition, insulin-like growth factor 1 (IGF-1) can prevent age-related deterioration of muscle function in mice, at least partially through activation of satellite cell proliferation (17-19). Furthermore, treatment of injured muscles with curcumin, which is a small-molecule inhibitor of the nuclear factor {kappa}B (NF-{kappa}B) transcription factor, markedly improves muscle regeneration (20). Additional candidates under investigation for ameliorating sarcopenia include prednisolone, androgens or estrogens, {beta}2-adrenergic agonists, and inhibitors of myostatin (GDF-8), a protein that inhibits skeletal muscle growth [reviewed in (21)].

It is possible that some or all of these treatments might work through a final common pathway to improve muscle repair or ameliorate sacropenia. For example, Notch and NF-{kappa}B may be members of a common pathway: A number of studies suggest that in at least some nonmuscle tissues, NF-{kappa}B affects the Notch signaling pathway as either a transcriptional activator of the Notch gene or as a gene that is targeted by activated Notch signaling (22-24). Further studies in skeletal muscle are needed to determine whether the improved muscle repair seen in response to Notch activation (5) and NF-{kappa}B inhibition (20) share common effectors. On the other hand, there is little evidence that IGF-1 signaling is NF-{kappa}B-dependent, suggesting that multiple pathways could lead to an improvement in muscle function.

A number of additional growth factors regulate embryonic myogenesis and repair of injured muscles in adult animals. Included among these are the fibroblast growth factors, hepatocyte growth factor, some interleukins, leukemia inhibitor factor, platelet-derived growth factors, and members of the transforming growth factor family, all of which affect muscle precursor cell proliferation [reviewed in (21)]. Furthermore, bone morphogenetic proteins (such as BMP4), hedgehog proteins, neuregulins, tumor necrosis factors, and members of the Wnt family affect muscle differentiation. Interaction of signaling pathways in muscle may be common. It is known, for example, that the BMP4 and Notch signaling pathways intersect through interaction of the BMP4 effector Smad1 with the intracellular domain of Notch, and there is also cross-talk between Wnt and Notch pathways through glycogen synthase kinase-3 (25, 26). Thus, it is likely that muscle regeneration is affected by synergistic or antisynergistic cross-talk between Notch and other signaling pathways.

In addition to the intrinsic properties of satellite cells, such as possible age-related changes in the expression of Delta, factors outside the muscle cells themselves may determine the success of muscle regeneration. For example, several types of cross-transplantation experiments suggest that old muscles can regenerate much better in a young host than in an old host [reviewed in (3, 4)]. Nonmuscle factors that could affect muscle function during aging or repair of injury include the extent to which the muscles are vascularized, successfully innervated by motor neurons, and resistive of connective tissue fibrosis (scarring) (3, 4, 27). Treatments that affect such nonmuscle factors also should be considered for their potential therapeutic effect on sarcopenia.

It now should be possible to begin to extend the findings of Conboy et al. from mouse models to initial human studies. For example, it should be possible to study Delta and Notch signaling in satellite cells obtained from biopsies of young and aged human muscles. If Delta up-regulation is impaired and Notch activation can be restored in cells from aged human muscles, then we could have confidence that findings in mice may be generalized to humans. Further development of Notch activation to improve repair in aged muscle, and possibly sarcopenia, could then proceed with a focus on animal studies and perhaps also human clinical trials.


December 3, 2003
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  29. Work in the authors' laboratories is supported by grants to J.B.M. from the National Institute of Arthritis and Musculoskeletal and Skin Diseases; the National Heart, Lung, and Blood Institute; the U.S. Department of Agriculture; and the Muscular Dystrophy Association; and to C.P.E. from the National Cancer Institute and the National Institute of Child Health and Human Development.
Citation: J. B. Miller, C. P. Emerson, Does the Road to Muscle Rejuvenation Go Through Notch? Sci. Aging Knowl. Environ. 2003 (48), pe34 (2003).








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