In order to compare muscle regeneration success and functional recovery between young (~20 year old) and aged (~70 year old) individuals, myofibre atrophy was induced by immobility (cast application for two weeks). This followed by acute exercise (loading) of skeletal muscle after cast removal (for 3 days and for 4 weeks), which aimed to promote muscle regeneration and functional improvement in strength and agility. Muscle biopsies were collected prior to immobility (basal level), after 2 weeks of immobility (induced atrophy), 3 days after cast removal (initiation of regeneration and functional recovery) and at 4 weeks after cast removal (ongoing regeneration and functional recovery). The scheme of this experimental setup is depicted in .
Immobility-induced muscle atrophy causes an age-specific increase in degeneration and lack of myogenic recovery
To determine whether muscle maintenance was age-dependent under the conditions of mobility, immobility-atrophy and loading-recovery, we analysed 10 µm cryosections derived from the young and old muscle biopsies at the indicated time points. As shown in , the muscle histology was markedly different between young and aged individuals, in the basal (‘Pre’) state (prior to immobility) and was particularly different during the immobility and recovery periods of immobility-induced atrophy. As compared to young, the old human muscle fibres were uneven in size and less numerous before immobility (Pre). Old myofibres underwent severe degeneration during immobility, as compared to mild degeneration of young myofibres (2 weeks). Additionally, old myofibres, but not young ones, exhibited a persistent inflammatory response and scar formation at both 3 days and 4 weeks of recovery (). The immobility-caused myofibre degeneration in old individuals was highly pronounced and similar to pathological degenerating muscle, with its typical clusters of new embryonic myosin heavy chain (eMyHC+
) myofibres and broken sarcolemma, evidenced by uneven pattern of dystrophin, e.g.
in cases of congenital myopathies (Renault et al, 2002
; Straub & Bushby, 2006
) (Fig S1A and B
of Supporting Information). In contrast, acutely deteriorating muscle clusters were absent, and intact dystrophin+
sarcolemma was typical in young muscles, thus suggesting better tissue maintenance during immobility (Fig S1
of Supporting Information). These data determine that both maintenance of immobilized skeletal muscle and regeneration of atrophic myofibres after cast removal, become inefficient in older individuals, manifested as the replacement of functional tissue by fibrotic scar tissue (Fig S1C–F
of Supporting Information).
In accordance with these findings, based on the quantification of quadricep muscle cross-sectional area (MRI), old muscle fibres were much smaller than young in the ‘pre’ state, and as compared to young, the size of old muscle fibres was not efficiently recovered, following cast removal and exercise (Fig S2A of Supporting Information). These observations are consistent with published literature and animal studies (Brown & Hasser, 1996
; Degens & Alway, 2003
; Machida & Booth, 2005
; Pistilli et al, 2007
). The histology and muscle size data were further confirmed and extrapolated by functional studies on muscle concentric/isometric strength and total muscle contraction work (Fig S2B–D
of Supporting Information)–establishing that while both young and aged individuals recovered close to basal levels of these functional parameters, by exercising after immobility, old muscles always remained weaker than young.
The age-specific decline in muscle fibre maintenance, repair, size and strength under all studied conditions (i.e.
during normal muscle use and during recovery from the immobility-induced atrophy) could result from known age-specific alterations in many parameters, such as innervation and vascularization, as well as the lack of muscle fibre regeneration (Grounds, 1998
; Thomas, 2001
; Wagers & Conboy, 2005
). Based on our work in the animal model, we hypothesized that a decline in the maintenance and repair of the muscle functional unit (myofibre maintenance via resident satellite cells) is a main factor causing the lack of old human muscle regeneration, strength and agility. Thus, we next examined whether the diminished size of aged myofibres, and their lack of regeneration, could be caused by an age-specific physical loss of muscle stem cells and/or by an age-specific decline in satellite cell activation.
Certain controversy exists in the published literature, with respect to the age-specific decline in numbers of satellite cells. Some studies report diminished numbers of these cells in older animals and humans, while other published data argues against such a decline (Collins et al, 2007
; Conboy et al, 2003
; Renault et al, 2002
; Schultz & Lipton, 1982
; Shefer et al, 2006
). Our data suggest that in mice, there is no significant decline in the number of quiescent satellite cells with age, but the ability of these cells to expand in response to traumatic muscle injury (induced by cardiotoxin or dry ice) declines due to the lack of Notch activation (Carlson et al, 2008a
; Conboy et al, 2003
). Consequentially, the numbers of satellite cells that are activated by injury to expand and produce proliferating fusion-competent myoblasts dramatically decline in old mouse muscle (Carlson & Conboy, 2007
; Carlson et al, 2008a
; Conboy et al, 2003
Different experimental systems and standards of what is considered to be a satellite cell are used in the field. Additionally, cardiotoxin and dry ice are not physiological agents of human muscle repair and remodelling. We therefore decided to clarify the situation by using our model of human myofibre regeneration after atrophy, and compare the number of quiescent muscle satellite cells associated with undamaged myofibres with the number of satellite cells activated by myofibre deterioration. Satellite cell numbers were quantified, using immuno-detection of three different markers: paired box gene 7 (Pax7), neural cell adhesion molecule (NCAM) and muscle-cadherin (M-Cadherin), in young and old human skeletal muscle cryosections.
As shown in , aging produces a ~2-fold decline in the number of satellite cells, endogenous to old muscle in the basal state. The number of old myogenic cells increases slightly during old muscle immobility (2 weeks), which is consistent with the ongoing degeneration and attempts at regeneration of old tissue, shown in and Fig S1
of Supporting Information. Importantly, there was a very pronounced, ~4-fold age-specific decline in the expansion of satellite cells in response to exercise after the immobility-induced atrophy (3 days and 4 weeks), . The age-specific decline in numbers of Pax7 myogenic cells during exercise after immobility was also confirmed by Western blotting (). These data establish that stem cell activation significantly declines with age in humans, which may contribute to the lack of muscle maintenance and repair, and to the replacement of myofibres by fibrous scar tissue in old people (, Fig S1
of Supporting Information).
Pronounced lack of myogenic cell expansion is detected in old human muscle that undergoes exercise after immobility
Previously, activation of Notch was determined to be indispensable for productive regeneration of young muscle, and capable of rescuing the repair of aged muscle in a mouse model of acute tissue injury (Carlson et al, 2008a
; Conboy et al, 2003
; Conboy & Rando, 2002
). Therefore, we set to determine whether (1) active Notch is present in young human satellite cells and becomes down-regulated in old satellite cells and (2) Notch ligand Delta is expressed at higher levels in young human regenerating muscle, as compared to old.
To establish whether Notch activation is lacking in old human satellite cells associated with aged muscle in vivo, we performed Notch and Pax7 co-immunodetection experiments in cryosections of human muscle biopsies. As shown in (and Fig S3 of Supporting Information), nuclear active Notch is eagerly detected in Pax7+ myofibre-associated cells. Furthermore, we also found that levels of the Notch ligand Delta are diminished in old myofibres, as compared to young myofibres (). In agreement with these data, decline in active Notch and its ligand Delta is observed in Western blot analysis of young and old human muscle ().
Notch activation and Delta upregulation is diminished in regenerating old human skeletal muscle
Notably, the expansion of myogenic cells in regenerating human muscle (during exercise after immobility) positively correlated with the levels of active Notch. The numbers of Pax7+
/Notch active cells were low in the basal state and during immobility, but greatly increased at 3 days post-immobility in young, but not in old human muscle (). The numbers of Pax7+
/Notch active human satellite cells declined after several weeks of regeneration, when the differentiation process typically follows initial cell expansion ( (Collins et al, 2005
; Wagers & Conboy, 2005
)). Accordingly, while there were still more Pax7+
/Notch active cells in the young, as compared to old human muscle at 4 weeks post-immobility, the total number of activated myogenic cells declined in both young and old tissue (). These results demonstrate that Notch regulation becomes altered during human aging in skeletal muscle, and suggests the importance of Notch for the expansion of human satellite cells.
To deepen the molecular understanding of the age-specific lack of organ stem cell responses in humans, we examined the activity of TGF-β/pSmad pathway in resting and regenerating young and old human skeletal muscle. In mice, the lack of Notch activation is compounded by an increase of TGF-β/pSmad3 that results in the regulation of CDK inhibitors, thus assuring loss of satellite cell regenerative capacity and deteriorated repair of old muscle (Carlson et al, 2008a
; Conboy et al, 2003
Remarkably, as shown in , these molecular signatures of aging within the muscle stem cell compartment are conserved between mouse and human that suggests the fundamental significance of uncovered regulatory mechanisms. As compared to young, old human muscle fibres contain higher levels of TGF-β, which associates with the laminin-rich basement membrane of the satellite cell microniche (). Accordingly, levels of nuclear pSmad3 (the transcriptional factor that is activated by TGF-β signalling) are excessive in old human satellite cells, as compared to youngs (). To further confirm these results in a more quantitative way, we also performed Western blot analysis of young and old human muscles. As shown in (quantified in ), the levels of TGF-β, pSmad3 and CDK inhibitors, p15 and p21 (known to be induced by TGF-β signalling and reduced by active Notch) are all higher in the old, as compared to young human muscle. Interestingly, p27 and p16 were undetectable in either young or old tissue, suggesting that these CDK inhibitors do not play a major role in studied processes. Efficient immuno-detection of p27 and p16 with the same antibodies was performed using positive control protein extracts (not shown).
Mechanisms of muscle stem cell aging are conserved between mice and humans, with respect to TGF-β signalling imbalance
To examine the effects of TGF-β on myogenic properties of human muscle stem cells, exogenous molecule was added to young and old human satellite cells in culture. Myogenic capacity was determined, based on number of fusion-competent, proliferating myoblasts, e.g.
cells that rapidly (in 2 h) incorporate bromodeoxyuridine (BrdU), co-express desmin and myogenic differentiation (MyoD) and fuse into eMyHC + myotubes when transferred to mitogen-low medium. As shown in and Fig S4 of Supporting Information, the myogenic regenerative potential was dramatically reduced by TGF-β1, thus confirming its role as a conserve between mouse and human negative regulator of muscle regeneration. Satellite cells isolated from young humans, exhibited higher myogenic potential as compared to the satellite cells derived from old people in these 24 h isochronic cultures, where the age of cells is matched with the age of sera (). An even higher magnitude of age-specific deficiency in myogenic responses was observed in 7 day isochronic human satellite cell cultures (Fig S5 and Table S1
of Supporting Information). Additionally, similar to findings in the mouse model, young human satellite cells had diminished regenerative responses, when cultured in the presence of old sera in heterochronic co-culture assays (Fig S5 and Table S1
of Supporting Information) (Carlson & Conboy, 2007
). Conversely, the myogenicity of old satellite cells was improved when cultured in young sera. These data are the first to demonstrate that cellular and molecular mechanisms of muscle stem cell aging are highly conserved between mouse and human, with respect to the age-specific decline in satellite cell activation and to the biochemical imbalance in TGF-β and Notch.
The molecular causes for the age-specific decline in Delta expression and Notch activation in mouse muscles remains unknown. However, it is well established that the expression of Delta and subsequent activation of Notch are positively regulated by MAPK during embryonic development of several distinct organs in Drosophila
and Caenorhabditis elegans
(Carmena et al, 2002
; Shaye & Greenwald, 2002
). Exploring the evolutionary and developmental conservation of Notch and MAPK cross-talk, we examined whether (1) MAPK pathway strength becomes diminished in old human muscles, as compared to youngs and (2) whether MAPK signalling intensity is causal for Notch activation and myogenic properties of human satellite cells. Quite interestingly, Western blot analysis of young and old human muscles, demonstrated that the MAPK signalling strength is indeed significantly down-regulated with age (). Extrapolating the functional significance of these findings, we examined the levels of Delta, amounts of active Notch and the efficiency of myogenic responses in human satellite cells cultured in the presence of agonists and antagonists of MAPK pathway. Remarkably, MAPK agonist fibroblast growth factor 2 (FGF-2) induced Delta and active Notch, while specific inhibitor of MAPK (MEK inhibitor) significantly attenuated Delta and active Notch levels (). As expected, the levels of pERK (a key downstream effector of MAPK) were induced by FGF-2 and reduced by MEK inhibitor, thus validating the success of experimental modulation of MAPK (). Furthermore, the myogenic regenerative capacity of young and importantly, old satellite cells was significantly enhanced through forced activation of MAPK, and even young satellite cells failed to produce proliferating fusion-competent myoblasts when MAPK was experimentally inhibited (, Fig S6 of Supporting Information). Consistent with the data shown above in control isochronic cultures, young satellite cells outperformed the old satellite cells ().
MAPK signalling strength becomes diminished in old human muscle, and is causal for Notch activation and myogenic properties of human satellite cells
To confirm that the main effect of MAPK on human satellite cell responses was through Notch activation, we activated MAPK in the presence of a Notch antagonist, gamma secretase inhibitor (GSI). As shown in Fig S7 of Supporting Information, inhibition of Notch by GSI precluded satellite cell regenerative responses even when MAPK was induced by FGF-2, thus suggesting that the positive regulation of satellite cell myogenicity by MAPK acts up-stream of Notch activation. These data establish that MAPK pathway is an age-responsive positive regulator of Notch in human muscle, and that the MAPK/Notch cross-talk is evolutionarily conserved between invertebrate embryogenesis and postnatal human muscle stem cell activation and aging.
Revealed cellular and molecular mechanisms of human muscle stem cell aging, prompted us to examine whether forced Notch activation would be able to restore myogenic responses to old human satellite cells cultured with aged human sera (old isochronic cultures). At the same time, we examined whether the regenerative responses of young human satellite cells cultured with young human sera would be incapacitated when Notch activation is inhibited (young isochronic cultures). Experimental activation of Notch receptor by exogenous ligand, Delta and forced inhibition of Notch by a GSI were performed for seven days of culture (control activation and inhibition of Notch shown in Fig S8 of Supporting Information; additional experiments on control young and old isochronic cultures are shown in Fig S5 of Supporting Information). Myogenic responses were measured, as described above, based on the generation of proliferating fusion-competent myoblasts. These experiments demonstrated that Notch is indeed a necessary and sufficient molecular determinant of human myogenic responses—required for productive myoblast generation by young stem cells, and capable of rescuing myogenesis of aged human satellite cells even in old systemic milieu (). To extrapolate these findings with higher molecular definition, we compared the levels of CDK inhibitors, p15 and p21 in satellite cells cultured in control isochronic conditions and those cells with forced activation and inhibition of Notch. The results of Western blotting shown in demonstrate that the levels of p15 and p21 are higher in old satellite cells as compared to young (in agreement with the age-specific elevation of these CDK inhibitors in human muscle in vivo, ). Importantly, Notch activation diminished the levels of these CDK inhibitors in human satellite cells, while Notch inhibition resulted in the up-regulation of p15 and p21 (). These data establish the functional significance of Notch activation for regeneration and maintenance of human muscle, and demonstrate that Notch is an important negative regulator of CDK inhibitors in human satellite cells.
Notch is a necessary and sufficient molecular determinant of human myogenic responses in vitro, which rescues productive regeneration in the presence of aged sera and attenuates expression levels of p15 and p21 in human satellite cell cultures