The Effect of Physiological Stimuli on Sarcopenia; Impact of Notch and Wnt Signaling on Impaired ...

30 Pages·2012·1.35 MB·English

Int. J. Biol. Sci. 2012, 8 731

Ivyspring
I nternational Publisher IInntteerrnnaattiioonnaall JJoouurrnnaall ooff BBiioollooggiiccaall SScciieenncceess
2012; 8(5):731-760. doi: 10.7150/ijbs.4262
Review
The Effect of Physiological Stimuli on Sarcopenia; Impact of Notch and Wnt
Signaling on Impaired Aged Skeletal Muscle Repair
Susan Tsivitse Arthur , Ian D. Cooley
Department of Kinesiology, Laboratory of Systems Physiology, University North Carolina - Charlotte, Charlotte, NC 28223,
USA.
 Corresponding author: Dr. Susan Tsivitse Arthur, Department of Kinesiology, UNC Charlotte, 9201 University City Blvd.,
Charlotte, NC 28223. Telephone: [phone] FAX: [phone] Email: [email protected].
© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/
licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.
Received: 2012.02.21; Accepted: 2012.04.06; Published: 2012.05.23
Abstract
The age-related loss of skeletal muscle mass and function that is associated with sarcopenia
can result in ultimate consequences such as decreased quality of life. The causes of sarcopenia
are multifactorial and include environmental and biological factors. The purpose of this review
is to synthesize what the literature reveals in regards to the cellular regulation of sarcopenia,
including impaired muscle regenerative capacity in the aged, and to discuss if physiological
stimuli have the potential to slow the loss of myogenic potential that is associated with
sarcopenia. In addition, this review article will discuss the effect of aging on Notch and Wnt
signaling, and whether physiological stimuli have the ability to restore Notch and Wnt sig-
naling resulting in rejuvenated aged muscle repair. The intention of this summary is to bring
awareness to the benefits of consistent physiological stimulus (exercise) to combating sar-
copenia as well as proclaiming the usefulness of contraction-induced injury models to studying
the effects of local and systemic influences on aged myogenic capability.
Key words: sarcopenia, physiological stimuli
Introduction
With aging there is a decline in the function of copenia-associated morbidity and mortality in the
multiple organ systems including heart, brain, nerves, aging population.
blood, skin and skeletal muscle. Sarcopenia is the A contributing factor to sarcopenia is a dimin-
age-associated loss of muscle mass and function, ished ability of aged muscle to repair itself following
which may appear as early as 50 years (y) in humans injury (4-8). There are multiple suggested mechanisms
(1). The prevalence of sarcopenia greatly increases for the impaired muscle regenerative process in aged
over the age of 80 y with reports of 50% or greater loss muscle including loss of satellite cell number or func-
of muscle strength (2, 3). tion, decreased myoblast proliferation or weakened
The causes of sarcopenia appear to be multifac- differentiation states. However, more studies are
torial and include hormones, sedentary lifestyle, needed before a definitive answer can be reached.
smoking, genetics, body size and composition. How- Although satellite cell activity is rigorous during
ever, the cellular aspects of age-associated sarcopenia muscle formation in the young, with age there is an
are not well elucidated. Because people are living attrition in satellite cell function (5, 7, 9-11). The novel
longer, understanding the mechanisms underlying finding that impaired Notch signaling may be partly
sarcopenia is critical to the development of therapeu- responsible for the loss of myogenic potential in aged
tic and prevention strategies that will decrease sar- muscle is intriguing, and provides a potential clue

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Int. J. Biol. Sci. 2012, 8 732
into the mechanisms underlying sarcopenia (7, 12, 13). etal muscle resulting in muscle atrophy. With the loss
In addition, there may be a dysfunctional orchestra- of muscle mass, there is an inability to perform activi-
tion of the signaling pathways, Notch and Wnt that ties of daily living which exacerbates the loss of
result in impaired muscle repair in the aged (14-17). strength, leading to disabilities, nursing home admis-
The coordination of these signaling pathways during sions and ultimately increased mortality (28). This
postnatal myogenesis is not well defined, nor is their review article will discuss the variety of manifesta-
contribution to impaired myogenic potential in aged tions associated with aging that cause sarcopenia.
muscle. Sarcopenia exerts its effects on other systems and in-
Physiological stimuli (exercise, injurious muscle clude, but are not limited to: cardiovascular, meta-
contractions, hypertrophy models) are known to in- bolic, and bone (1, 29, 30). Since skeletal muscle is a
crease Notch and Wnt signaling in young muscle component of cardiorespiratory fitness,
(18-23). However, little is known about the influence age-associated skeletal muscle atrophy results in poor
of physiological stimuli on the expression and coor- oxyten uptake by skeletal muscle and sedentary life-
dination of Notch and Wnt signaling in repairing style weakens cardiovascular function. In addition,
aged muscle. Studying the effect of physiological there is impaired oxidative metabolism in atrophic
stimuli on Notch and Wnt coordination during the skeletal muscle, resulting in poor glucose regulation
regenerative process of aged muscle may lead to an and metabolic disease. Abnormal gait and disabilities
effective interventional strategy for sarcopenia. Con- ensues with muscle atrophy and sedentary lifestyle as
sequently, such studies deserve further exploration. well as the bone disease osteoporosis (mus-
The purpose of this review is to examine the cellular cle-associated force applied to the bone is needed for
regulation of sarcopenia, including impaired muscle good health fitness) (1, 29, 30). Sarcopenia negatively
regenerative capacity in the aged, and to discuss if influence other organs whose destructive outcomes
physiological stimuli have the potential to slow the result in loss of independence, and ultimately in-
loss of myogenic potential that is associated with creased morbidity (1, 30).
sarcopenia. In addition, this review article will discuss
Causes of sarcopenia
the effect of aging on Notch and Wnt signaling, and
whether physiological stimuli have the ability to re- There are a variety of factors that contribute to
store Notch and Wnt signaling resulting in rejuve- sarcopenia, including both environmental and bio-
nated aged muscle repair. A better understanding of logical factors. For example, poor nutrition and a
these issues may lead to novel therapeutic strategies sedentary lifestyle are contributors to sarcopenia (2,
to prevent the loss of muscle quality seen in sarcope- 29). The loss of appetite that afflicts many elderly in-
nia. dividuals leads to decreased food intake and Vitamin
D and protein deficiencies. This may impair protein
Impact of sarcopenia
synthesis and ultimately decrease muscle mass (2, 29).
Sarcopenia (also called senile muscle atrophy) is Edstrom et al. suggest that dietary intervention may
an age-related loss of skeletal muscle mass and func- be a tool to combat sarcopenia (26). In addition to loss
tion that is often determined by measuring the skele- of appetite, many elderly individuals suffer from or-
tal mass index (24). The percentage of skeletal muscle thopedic pathologies, and associate movement with
index is calculated by dividing muscle mass by the pain, resulting in decreased physical activity and ul-
squared height, or by dividing muscle mass by body timately loss of muscle mass and strength (30). How-
mass (24, 25). Sarcopenia is experienced primarily in ever, elderly individuals who are highly active (such
the lower extremities, which may be related to re- as Masters Athletes) also have significantly less mus-
duced physical activity with age, or to greater loss of cle mass than their younger counterparts, suggesting
motor units in the legs than arms (25, 26). Evidence that disuse may not be the sole contributor to sarco-
suggests that the age-related loss of muscle mass is penia (30, 31). Thus, other age-related causes of sar-
the cause of poor strength, yet the impairment of copenia, besides an increased sedentary lifestyle, need
muscle strength and power (dynapenia) is more pro- to be delineated (25). Although lifestyle is an im-
found than the loss of muscle mass (27). portant player in the development of sarcopenia, the
There is a dichotomy in the relationship between majority of contributors to age-related loss of muscle
age-associated mortality and sarcopenia. The negative efficiency involve multiple biological systems such as:
attributes associated with aging including poor nutri- central and peripheral nervous systems, endocrine,
tion, sedentary lifestyle, pain associated with range of immune, and metabolic systems (8, 25, 28-30, 32, 33).
motion, as well as the degenerative cellular manifes- This review will discuss what are thought to be the
tations cause an inability to use the vital organ, skel- major contributing factors underlying the physiolog-

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Int. J. Biol. Sci. 2012, 8 733
ical manifestations of sarcopenia. Pro-inflammatory cytokines and adipokines deter
muscle mass formation and promote fat mass accu-
I. Molecular and Cellular Manifestations asso-
mulation (28, 29). Elevated TNFα in aged muscle is
ciated with Sarcopenia
associated with decreased muscle force production
a. Increased intramuscular fibrosis and adipose (44, 45). TNFα is also linked to sarcopenia because this
tissue: The tissue composition of sarcopenic skeletal pro-inflammatory cytokine is known to be associated
muscle is altered and consists predominately of con- with other factors that contribute to sarcopenia in-
nective and adipose tissue, a condition termed my- cluding protein degradation, reactive oxygen species
osteatosis (25, 28). In obese aged individuals, this oc- (ROS) accumulation and apoptosis (35, 46). In addi-
currence is termed “Sarcopenic Obesity” (25, 28). In- tion, TNFα may be associated with sarcopenia by
creased fibrosis within the sarcopenic muscle may be promoting insulin resistance, delaying muscle repair,
related to elevated extracellular matrix protein (col- and exacerbating the pro-inflammatory response by
lagen) levels, as well as the accumulation of debris up-regulating IL-6 (25, 43, 45-47).
from impaired protein degradation (14, 26, 34). In Because IL-6 has both pro- and an-
addition, there is greater fibronectin expression in ti-inflammatory characteristics and has effects on
aged myofiber explants compared to young myofiber muscle growth and atrophy, it is difficult to discern
explants (14). the role of IL-6 in the development of sarcopenia.
b. Increased pro-inflammatory cytokines TNFα There is a negative correlation between IL-6 and skel-
and IL-6: Aging is associated with a state of chronic, etal muscle strength in the elderly, and
low inflammation. There are many reports of over-expression of IL-6 is associated with muscle at-
increased levels of the pro-inflammatory cytokines rophy (48, 49) IL-6 may contribute to insulin re-
tumor necrosis factor α (TNFα) and interleukin- 6 sistance and inhibit insulin-like growth factor-1
(IL-6) in the systemic circulation of the elderly (35-42). (IGF-1), which promotes protein degradation during
For example, there was a 2.8 fold increase in TNFα sarcopenia (47, 50). Inhibiting IL-6 with an antibody
expression in skeletal muscle of aged (~ 70 y) male or an anti-inflammatory reagent results in increased
subjects compared to young (~20 y) male subjects (38). protein synthesis and a rescue of the loss of muscle
Phillips et al. also reported increased expression of mass (51, 52). Additional research is needed to delin-
TNFα in soleus and vastus lateralis (VL) of aged (26 eate the relationship and contribution of TNFα and
month (mo)) rats relative to young (6 mo) rats (39). IL-6 to sarcopenia.
Furthermore, centurions were found to have signifi- c. Decreased sex hormones: As one ages, there is
cantly higher plasma TNFα levels than younger (18 – a direct correlation between the levels of sex
30 y) controls with corresponding elevations of IL-6 hormones and muscle mass suggesting that depletion
(37). Studies report a link between elevated plasma of testosterone and estrogen may contribute to
IL-6 with age and increased mortality (40-42). Rou- sarcopenia (1, 8). In addition, it is suggested that the
benoff et al. reported increased plasma levels of IL-6 in age-associated decline in estrogen and testosterone
aged (~ 79 y) subjects relative to young (~ 39 y) con- are related to increases in levels of the
trols. However, there was no difference in plasma pro-inflammatory cytokines IL-6 and TNF, which
TNFα levels between the age groups (42). High levels may accelerate the loss of muscle mass during sarco-
of IL-6 and TNFα are associated with a multitude of penia (8, 53, 54). With aging, there is also a correlation
age-related diseases including obesity, cardiovascular between decreased sex hormone levels and a decline
diseases, type II diabetes and sarcopenia (35, 36, 43). It in the growth factors of growth hormone (GH) and
should be noted however, that some reports have not IGF-1, which may contribute to sarcopenia (54, 55).
found differences in plasma and skeletal muscle Postmenopausal (58-70 y) women possess lower GH
TNFα or IL-6 levels between young and aged models; levels than premenopausal (45-51 y) women, and the
but rather suggest that the aged environment may be lack of GH is known to promote intramuscular fat
more sensitive to the effects of these accumulation and loss of muscle mass (8, 55). Fer-
pro-inflammatory cytokines (36). rando et al. report that aged (> 60 y) men who were
Although the mechanism for the potential eleva- administered testosterone therapy showed increased
tion of TNFα and IL-6 with age, and the relationship IGF-1 protein levels (56).
of these cytokines to sarcopenia are not well defined, Between the age ranges of young (20 – 29 y) and
they may be related to increased levels of adipose aged (70 – 84 y), bioavailable testosterone drops ~ 4.2
tissue in the elderly (1, 30). Adipocytes secrete IL-6 fold and the testosterone precursor dehydroepi-
and TNFα as well as the adipokines leptin and adi- androsterone (DHEA) falls ~ 67 fold (57). Between the
ponectin, which promote inflammation. ages of 70 and 102 years, testosterone levels decrease

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Int. J. Biol. Sci. 2012, 8 734
with a corresponding decline in muscle strength, and age-associated loss of sex hormones contributes to
with testosterone supplementation, there is a rescue of sarcopenia directly, but also influences other factors
muscle mass and function (56-60). Ferrando et al. re- that exacerbate the loss of muscle mass and function
ported that aged (> 60 y) male subjects who were and the accumulation of intramuscular adipose tissue.
administered testosterone supplementation for 6 mo Thus although the decline of sex hormone levels with
experienced gains in leg and arm muscle strength as aging contributes to sarcopenia, straightforward re-
well as increased muscle protein synthesis (56). Sin- placement of under-produced estrogen or testos-
ha-Hikim et al. reported similar findings in which terone may not be the most successful treatment
aged (60-75 y) males who were administered testos- strategy because these hormones also interact with
terone enanthate for 20 weeks (wk) displayed in- other systems (inflammation, growth factors).
creased cross sectional area of VL muscle and in- d. Alterations to muscle fibers and motor units:
creased expression of markers of myogenicity (58). Both muscle fiber loss and decrease in skeletal muscle
Furthermore, aged (22 mo) mice given a testosterone cross-sectional area are associated with sarcopenia
implant for 2 mo experienced diminished oxidative (66). There is a switch of muscle fiber types from type
stress and myostatin levels, as well as increased my- II to type I, and a greater loss of type II muscle fibers
ogenicity (60). These data show that testosterone cor- than type I in aged skeletal muscle (25, 31, 67, 68). The
relates with sarcopenia, muscle mass and function as VL of aged (~ 68 y) men had a higher percentage of
well protein synthesis function. Consequently, re- fibers expressing Myosin heavy chain (MHC) type I (~
storing testosterone levels in aged skeletal muscle 20%) relative to VL of young (~ 28 y) subjects (~ 8%)
may be favorable for the prevention or reversal of (31). In addition to the loss of muscle fibers is an
sarcopenia. age-associated remodeling of the muscle architecture
The decline in expression of estrogen in women in which the muscle fiber fascicle length and the in-
that contributes to menopause (average age of onset is sertion angle decreases and there is a loss of sarco-
51.4 y) (57) is associated with sarcopenia (8, 61). Loss meres (69). Multiple mechanisms appear to underlie
of estrogen may promote body composition changes, the structural alteration of the skeletal muscle archi-
including a loss of muscle mass, but an increase in tecture, including a decreased rate of protein synthe-
adipose tissue as well as a redistribution of body fat to sis within the aged muscle. These changes ultimately
the visceral region (54, 61, 62). Maltais et al. suggests result in decreased aged muscle force production (25).
that estrogen may prevent fat accumulation within Motor unit alterations may be a contributing
skeletal muscle and may have a direct relationship factor to the age-associated loss of Type II muscle fi-
with lipoprotein lipase (which catalyzes triglyceride bers (70). These motor unit alterations include a de-
utilization). Thus, increased intramuscular fat in crease in firing frequency and motor unit recruitment,
postmenopausal women may be related in part to loss of Type II motor units and a reduced number of
deceased estrogen levels. Decreased muscle strength motoneurons innervating muscle fibers, especially
correlates with the age-associated loss of estrogen Type II muscle fibers which are then more susceptible
(54). Replacing estrogen levels with hormone re- to muscle atrophy and possibly sarcopenia (66, 71-73).
placement therapy (HRT) to increase muscle mass and Accelerated denervation may be related to elevated
strength is controversial (61, 63). Some reports show oxidative stress and accumulation of dysfunctional
that HRT increases muscle mass and strength (61, 62). protein machinery at the neuromuscular junction
Taking HRT for one year resulted in increased quad- within aged skeletal muscle (74). Using electron mi-
riceps cross sectional area (6.3%) and decreased croscopy images to study the neuromuscular junction
quadriceps intramuscular fat (4.9%) (61). However, (NMJ) structure, superoxide dismutase knockout
there are studies that suggest HRT may not be an transgenic mice displayed greater NMJ degeneration
ideal tool for sarcopenia as there were no changes in with dysfunctional mitochondrial activity in close
muscle mass or strength with HRT and there are re- proximity as well as decreased contractile force pro-
ports of significant negative consequences associated duction relative to wild type, suggesting impaired
with HRT such as increased risks of cancer and car- neuromuscular junction contributes to sarcopenia
diovascular diseases including stroke (54, 63-65). (75). In addition, there is decreased presence of ciliary
Rossouw et al. report a 26% and 37% increase in inva- neurotrophic factor and vascular endothelial growth
sive breast cancer and colorectal cancer respectively factor, both of which are important for motorneuron
(65). It is interesting to note that women over the age stability (25). Remaining denervated muscle fibers
of 65 who were on estrogen replacement therapy for attempt to compensate by re-innervating the surviv-
three years did not experience any changes in body fat ing motor units, which consequently change the fiber
percentage or physical performance (64). The type to slow-twitch (Type I). However the accelerated

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Int. J. Biol. Sci. 2012, 8 735
rate of denervation is faster than the decreased rate of IGF-1 and the cytokines TNFα and IL-6 are considered
reinnervation, resulting in loss of muscle function (25, indicators for sarcopenia (83).
30, 66, 73, 75). Some suggest that degeneration within f. Decreased IGF-1 and mRNA translation: The
the neuromuscular system may be the greatest con- decreased presence of IGF-1 in aged skeletal muscle
tributing factor to sarcopenia, while others contend may contribute to sarcopenia by severely impacting
that the muscle fiber denervation plays only a minor protein synthesis. IGF-1 has anabolic effects on mus-
role (26, 76). Edstrom et al. reports that there is insig- cle protein content by inhibiting protein degradation
nificant motorneuron loss during aging and suggests and promoting myogenesis. Thus, IGF-1 attrition in
that impaired reinnervation of the myofiber is a cause aged skeletal muscle is associated with less protein
of poor muscle regenerative response and sarcopenia synthesis and muscle growth (84, 85). In the presence
rather than motorneuron denervation (Edstrom #26). of IGF-1, aged (25 mo) rats increased total protein
Although correlations of motorneuron degeneration content by 27% in gastrconemius muscle relative to
and sarcopenia are reported, more research is needed control (84). In addition, there was increased muscle
to provide compelling evidence that motorneuron mass and force production in mdx mice (mouse model
degeneration is a major player in the development of that represents Duchenne muscular dystrophy) that
sarcopenia. Potential studies could include testing if were treated with IGF-1 (86, 87). Injection of an
reinnervation is the limiting factor by comparing adeno-virus with overexpressed IGF-IEa into aged (27
markers of sarcopenia in denervated muscle that was mo) mice resulted in increased muscle mass and
exposed to the microenvironment that promotes strength (88). IGF-1 administration to the aged skele-
reinnervation or a microniche that inhibits reinnerva- tal muscle may negate the impaired protein synthesis
tion. observed in sarcopenia.
e. Protein synthesis/degradation imbalance: A A reduction in IGF-1 may also influence protein
disruption of the balance between protein synthesis synthesis in aged skeletal muscle via inefficient
and protein degradation, resulting in the accumula- mRNA translation (79, 89-92). The mammalian target
tion of damaged proteins, is also associated with ag- of rapamycin (mTOR) signaling pathway is important
ing skeletal muscle and sarcopenia (1, 77, 78). There is for translation initiation and is therefore critical for
increased protein modification with aging possibly muscle protein synthesis. One mechanism that acti-
related to oxidative stress (78). Exacerbating the vates mTOR signaling is the
problem is the fact that the primary pathways used to IGF-1/PI3k/serine/threonine kinase (Akt) pathway.
remove modified proteins (proteasome system, au- Downstream effectors of mTOR signaling include
tophagy and lysosomal degradation) are less active in 70-kilodalton ribosomal S6 protein kinase (p70s6k),
aged muscle (1, 78, 79). For example, in the lysosomal eukaryotic translation initiation factor 4E binding
pathway in aged muscle, there is diminished delivery protein 1 (4E-BP-1), eukaryotic translation initiation
of membrane proteins or organelles to the lysosome factor 4E (eIF-4E), and ribosomal protein S6 kinase
and fusion of vacuoles with lysosomes (79). The attri- (S6K1). Aged rodent muscles have reduced mTOR
tion of protein degradation pathways results in (Akt phosphorylation) and p70s6k signaling (89-93).
accumulation of damaged proteins. Paturi et al. reported that aged (36 mo) mouse soleus
The rate of protein synthesis in aged muscle is had less Akt (and phosphorylated Akt), p70s6k and
reduced, with reports of decreased myofibrillar pro- phosphorylated p70s6k), phosphorylated mTOR, S6
tein content and MHC expression (77, 78). There are ribosomal protein, and AMP activated kinase (AMPK)
multiple causes of decreased protein synthesis in aged than young (6 mo) mouse soleus (91). With under-
skeletal muscle, such as underproduction of circulat- production of IGF-1 in aged skeletal muscle, there
ing and tissue-associated GH and IGF-1 isoforms (80, may be impaired mTOR signaling and mRNA trans-
81). Low levels of IGF-1 in aged muscle is related to lation, thereby imposing a barrier to protein synthesis
high expression of muscle growth inhibitors including in aged muscle.
the pro-inflammatory cytokines TNF and IL-6 (2, 38, g. Increased myostatin: Additionally, decreased
73, 82). In skeletal muscle of aged (70 y) subjects, there protein synthesis during sarcopenia may be a result of
is a 45% decrease in growth hormone receptor protein elevated myostatin expression in aged skeletal muscle
(GHR) and IGF-1 mRNA, along with a 2.8 fold in- (32, 73, 94, 95). There is a two-fold increase in myo-
crease in TNF mRNA relative to young (20 y) sub- statin expression within skeletal muscle of aged (70 y)
jects (38). Aged women with lower IGF-1 levels and subjects relative to young (20 y) subjects (38). GH and
greater IL-6 expression relative to younger counter- IGF-1 may have an inhibitory effect on myostatin,
parts, demonstrate aspects of disability and increased therefore one potential cause of increased myostatin
mortality (28, 83). This opposing relationship between in aged skeletal muscle is the attrition of GH and

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Int. J. Biol. Sci. 2012, 8 736
IGF-1 expression (38, 73, 95, 96). Myostatin may in- muscle repair (miR 221 and -181a) are decreased in
hibit muscle growth by preventing satellite cell acti- aged (24 mo) mice quadriceps (109), suggesting that
vation, as well as promoting an adipogenic cell fate these skeletal muscle miRNAs may be associated with
over myogenicity (95, 97). Inhibiting myostatin may the decreased muscle growth in aged muscle. More
be a key therapeutic target for sarcopenia. Admin- research is needed to determine the influence of
istration of myostatin inhibitors to aged (18 mo) mice miRNAs on sarcopenia.
resulted in a 12% increase in fiber cross-sectional area i. Apoptosis: Apoptosis of myofiber myonuclei
and 35% increase in force production by the tibialis and satellite cell nuclei within aging skeletal muscle
anterior (TA) (94). By understanding the mechanisms may result in muscle mass attrition that is associated
of the disruption in the pathways for protein degra- with sarcopenia (33, 111, 112). Apoptosis is a pro-
dation and protein synthesis during sarcopenia, grammed death of a cell or nucleus, which is activated
therapeutic interventions can be devised to rejuvenate by either extrinsic or intrinsic cues such as activation
muscle growth and remove damaged muscle proteins of death receptors, reactive oxygen species (ROS) ac-
in aged muscle. cumulation, endoplasmic reticulum stress or mito-
h. Altered microRNA expression: MicroRNAs chondrial stress. Upon activation, components of
(miRNA; miR) are novel post-transcriptional regula- apoptosis signaling are elevated including caspase 3,
tors that may contribute to age-related impairments cytochrome c, BCL-2 associate-X protein (Bax), apop-
by dysregulating gene expression, either by cleaving tosis-inducing factor (AIF), and/or apoptosis activat-
mRNA or by inhibiting translation (98-100). Recent ing factors (APAF-1) (33, 111, 112). The end result of
evidence suggests that miRNAs play a role in myo- myonucleus or cellular apopotsis is DNA fragmenta-
genesis (98, 101-107). During human muscle devel- tion and destruction of the cell, resulting in the for-
opment there is increased expression of muscle spe- mation of apoptotic bodies (33, 111-113).
cific miRNAs including miR-1,-133a, and -206 (103). Aged skeletal muscle has increased expression of
During postnatal myogenesis, these specific miRs pro-apoptotic proteins and caspases and DNA frag-
may play a role in skeletal muscle repair by influenc- mentation, with a concomitant decrease in expression
ing myoblast proliferation and differentiation (98, of anti-apoptotic proteins (112-119). There are eleva-
101-108). One week after injecting a mixture of miR-1, tions in APAF-1, Bax levels and caspase-3 activity
- 206 and - 133 into injured muscle, there was acceler- (113, 117). Relative to adult (12 mo) rat gastrocnemius,
ated muscle repair (104). miR-133a promotes myoblast aged (26 mo) rat gastrocnemius expressed higher lev-
proliferation by repressing factors required for muscle els of the pro-apoptotic protein AIF, with a concomi-
differentiation such as serum response factor (SRF), tant decrease in the apoptotic inhibitor known as the
myogenin and MHC (107). miR-1 contributes to my- apoptotic repressor with a caspase recruitment do-
oblast differentiation by inhibiting myocyte-specific main (ARC) (120). There are reports of increased an-
enhancer factor 2C transcriptional repressor, histone ti-apoptotic proteins including XIAP in aged skeletal
deacetylast 4 (HDAC4). miR-206 also contributes to muscle, which are thought to assist in counteracting
myoblast differentiation possibly by inhibiting Pax 7 the pro-apoptotic environment within the aged mus-
(101, 106, 107). Inhibiting miR-1 and miR 206 delays cle. However, the increased anti-apoptotic attempts
myoblast differentiation and there is a concomitant may be futile because there are also elevations in a
increase in Pax 3 expression (102). pro-apoptotic protein that inhibits XIAP
Because it has recently been determined that (Smac/DIABLO) (33, 121). If apoptosis does indeed
miRNA are important to myogenesis, it would be of play a role in sarcopenia, then research directed to-
interest to characterize miRNA in aged skeletal mus- wards decreasing DNA fragmentation and caspases
cle. There is a correlation between miRNA expression and promoting anti-apoptotic protein expression in
and aging that may link miRNAs to sarcopenia (99, aged skeletal muscle is warranted.
109, 110). Drummond et al. profiled miRNAs in VL j. Telomere shortening: With aging there is cel-
aged (~ 73 y) men relative to young (~ 31 y) male lular senescence in multiple organs. If this process
subjects and observed that miRNAs let-7b and let-7e occurs in skeletal muscle, then it may contribute to
(from the let-7 family) were elevated in aged muscle sarcopenia (4, 122). The “Hayflick Limit”, defined as
relative to young muscle. Let-7 family miRNAs are the limited number of cell replications prior to cell
related to decreased cell cycle regulators during mus- cycle arrest, may be a cause of cell senescence (123).
cle repair, so the authors suggest that decreased mus- Telomere instability may induce cellular arrest and
cle repair mechanisms in aged muscle may be a result limit cell replications (124). Telomeres are repeated
of elevated Let-7 family miRNA in aged muscle (110). DNA sequences that form a T loop cap at the end of a
The expression of miRNAs associated with skeletal linear chromosome and protect the chromosome ends

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Int. J. Biol. Sci. 2012, 8 737
from degradation. Addition of DNA sequences to meres within hematopoietic stem cells (HSCs) result
telomeres occurs in the presence of telomerase (4). in dysfunctional HSCs and increased HSC senescence
With every round of cell division the telomeres (128, 129). There is impaired activation and prolifera-
shorten and telomerase activity decreases. With in- tion of HSCs with shortened telomeres (128, 129).
creasing age, cells reach the maximum number of cell Since telomere shortening has been shown to impair
divisions. At this point, telomere length reaches a stem cell function, it is plausible to consider that te-
critical length and the chromosome may become un- lomere shortening occurs within cells of aged skeletal
capped. This in turn may result in disruption of muscle, including the adult skeletal muscle stem cells
chromosome integrity because uncapped telomeres termed satellite cells. The destructive outcome of te-
activate signaling pathways that correlate with DNA lomere shortening in aged skeletal muscle cells may
damage and apoptosis (Figure 1) (124). contribute to sarcopenia, but more research is needed.
Telomere shortening is used as a marker for ag- However, it should be noted that investigators have
ing, and it is recognized that telomere shortening in- found that satellite cells from aged skeletal muscle
duces premature aging and renders an organism maintained their telomerase activity although to a
susceptible to disease (122, 125). Mice with deficient lesser degree than those from young skeletal muscle.
telomerase activity exhibit shortened telomeres and This suggests that aged skeletal muscle stem cells
have a decreased lifespan (126, 127). There is also a have the ability to maintain their myogenic potential
relationship between telomere shortening and senes- (130).
cence of aged stem cells (128, 129). Shortened telo-

Figure 1. Telomere function and attrition with aging.

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Int. J. Biol. Sci. 2012, 8 738

k. Oxidative Stress: Accumulation of reactive copenia is an impaired ability of aged skeletal muscle
oxygen species (ROS), known as oxidative stress to regenerate following exposure to injury.
within aging skeletal muscle contributes to sarcopenia
II. Impaired Muscle Regeneration as a Con-
(1, 4, 131, 132). There are multiple sources of ROS
tributor to Sarcopenia
production within aged skeletal muscle (30, 132-134).
Lifelong accumulation of mitochondrial DNA With aging there is a delay in healing of tissue
(mtDNA) mutations in tissues that undergo high ox- when exposed to injury as evidenced by poor skin
idative phosphorylation such as skeletal muscle, re- healing, delayed bone repair, inability to re-myelinate
sults in decreased electron transport chain activity, damaged axons, impaired angiogenesis and delayed
impaired oxidative phosphorylation and ROS accu- colonic mucosal repair (138-140). There is also a defi-
mulation (30, 132, 133). Pro-inflammatory cytokines cient regenerative response in aged skeletal muscle
such as TNFα, which have been shown to be elevated following exposure to injury (4-8). While there was
in aging skeletal muscle, also promote ROS accumu- complete recovery of muscle mass at 21 days post
lation (134). chemical-induced muscle injury in 3 mo old rat tibialis
ROS (as well as reactive nitrogen species) accu- anterior muscle, there was 40% loss of muscle mass in
mulation within aged skeletal muscle causes tissue 31 mo old rat tibialis anterior muscle at the same time
degradation, skeletal muscle atrophy, decreased point (6). The ability to reinnervate and revascularize
muscle function and increased presence of fibrotic is reduced in aged muscle compared to young muscle
tissue (1, 4, 30, 131-134). Oxidative stress may also (141-143). There was significant motor denervation,
induce DNA damage and impair the ability of DNA including loss of motor axon terminals in the gas-
polymerases to copy strands, resulting in telomere trocnemius of aged (32 mo) mice (142). Using an in
shortening (122). The end result of oxidative stress is situ blood vessel growth protocol (corneal mi-
decreased longevity, and this is exacerbated by cropocket assay), Smythe et al. reported that aged (21
age-associated attrition of antioxidants and heat shock mo) rat muscle exhibited less blood vessel growth
protein levels (8, 30, 131). It would seem reasonable than young (3 mo) rat muscle (141). In addition to
that inhibiting ROS accumulation would increase poor reinnervation and revascularization, the muscle
longevity (135). In transgenic mice that over-express contraction apparatus is affected during recovery of
the antioxidant catalase, there is decreased production aged muscle (144) Lorenzon et al. reported no excita-
of ROS and hydrogen peroxide and fewer mitochon- tion-contraction (e-c) coupling mechanism present in
drial deletions resulting in a 21% increase in lifespan primary culture myoblasts obtained from aged hu-
relative to control (135). However, many researchers man muscle biopsies, while young primary culture
report that inhibiting ROS does not prolong lifespan myoblasts demonstrated e-c coupling at day 6 of dif-
(123, 136, 137). Transgenic mice that over-express the ferentiation (144). The delay in skeletal muscle repair
antioxidant, superoxide dismutase did not have an following injury (chemical-induced, exercise-induced)
extended lifespan (137). Furthermore, transgenic mice may be a contributing factor to sarcopenia. Below is a
that over-expressed two antioxidants; superoxide brief discussion of causes of impaired repair of aged
dismutase and catalase, did not display an extended skeletal muscle.
lifespan (136). Because of the age-associated accumu- a. Cause of impaired aged skeletal muscle repair
lation of mtDNA mutations, elevated – resident satellite cell number: A dysregulation of
pro-inflammatory levels and depressed antioxidant satellite cells could result in impaired repair of aged
expressions, there is a robust presence of oxidative muscle. Muscle regeneration was compared between
stress within aged skeletal muscle. The propensity for young (20 y) and aged (70 y) human subjects who
ROS accumulation within aged skeletal muscle may were exposed to 2 wk muscle cast immobilization
induce DNA damage, deplete energy stores and ex- followed by 3 day or 4 week re-loading (cast removal)
acerbate apoptosis, thereby contributing to sarecope- (5). During re-loading there was a ~ four - fold de-
nia (111, 132). Devising methods to decrease ROS ac- crease in satellite cell activation in aged muscle rela-
cumulation within aged skeletal muscle may be a tive to young muscle, suggesting that aged muscle
promising interventional strategy for sarcopenia. may possess dysfunctional satellite cells (5). It is un-
There are multiple contributors to sarcopenia clear if the contributing role of satellite cells to delin-
that are associated with DNA damage, apoptosis and quent aged muscle regeneration is related to changes
protein degradation. More research is needed to fur- in resident satellite cell numbers, or to their ability to
ther delineate these molecular manifestations of ag- function. It is difficult to determine if the overall
ing, and to devise strategies to counteract these dele- number of satellite cells in aged muscle is a contrib-
terious changes. Another contributing factor to sar- uting factor to poor repair because there is con-

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Int. J. Biol. Sci. 2012, 8 739
founding evidence regarding changes in satellite cell to activate and proliferate resulting in a delay of my-
number with age. Some investigators report a de- otube formation (7, 9).
crease in satellite cell number with age, while others However, there is evidence that the differentia-
suggest no change or an increase in resident satellite tion component of the myogenic program is defective
cell numbers in aged skeletal muscle (5, 7-9, 32, 73, in aged muscle (10, 11, 13, 30, 151, 153). Although
139, 145-149). Using Pax 7, neural cell adhesion there was no significant difference in the percentage
marker (NCAM) and MCadherin satellite cell mark- of desmin-positive cells between myoblasts obtained
ers, Carlson et al. reported a decrease in satellite cell from young (~ 30y) and aged (~83y) human myofiber
number in resting aged (~ 70 y) relative to young (20 explants, there was a significant decrease in the fusion
y) human muscle (5). Using a mouse model, Shefer et index of aged myotubes relative to young myotubes
al. showed a 60% decrease in Pax 7 positive-cells of (11). The authors also reported that the aged myo-
aged (19 – 25 mo) relative to young (3 – 6 mo) EDL tubes had fewer myonuclei than young myotubes,
myofibers (9). A plausible explanation for the per- and that the aged myotubes morphology appeared
ceived decrease of resident satellite cell number with meager (11). Lees et al. reported that although there
age is that satellite cells may have reached their was no difference in the profile of proliferating pro-
“Hayflick limit” in which they have reached the teins from cultured myogenic precursor cells (mpcs)
maximum threshold of cell divisions. Although there between young (3 mo) and aged (32 mo) rat muscle,
is a preponderance of evidence supporting the con- the aged mpcs expressed 50% less of the differentia-
cept of decreased satellite cell number with aging, it tion proteins myogenin and creatine kinase relative to
should be noted, however, that there are reports stat- young mpcs. In addition, there was less expression of
ing no differences in satellite cell number with age (11, p27kip1, and FOXO1 (proteins essential for myoblast
150). Using electron microscopy, dystrophin-laminin differentiation) in the aged relative to young mpcs
immunohistochemistry, and MyoD immunohisto- (151).
chemistry techniques for quantifying satellite cells, Altered expression of myogenic regulatory fac-
Brooks et al. did not observe any significant differ- tors (MRFs) and myostatin may play a role in inhib-
ences in satellite cell numbers between young (5 mo) iting proliferation or differentiation in aged regener-
and aged (24 mo) Fisher rat soleus muscles (150). ating muscle. Expression of MyoD, myf5, myogenin
b. Cause of impaired aged skeletal muscle repair and MHC are decreased in aged muscle (30, 73). In
– altered myogenic program: Independent of the con- addition, there may be decreased protein turnover in
tribution of resident satellite cell number, it is well aged muscle resulting in impaired myosin replace-
known that there is a problem with satellite cell func- ment (30). Furthermore, the presence of free radicals
tion in the myogenic program of aged muscle (7, 9-11, in aged muscle renders the myosin molecule suscep-
151). However, exactly what aspect of the myogenic tible to oxidation and weakens cross bridge formation
program is defective in aged muscle remains contro- (30, 154). There is also decreased expression of
versial. Some investigators state that myoblast prolif- E-proteins, which are critical co-regulators of MRFS,
eration is the limiting factor for effective age muscle but increased expression of Id, an inhibitor of differ-
repair, while others suggest that the impairment is entiation (30, 155). Id promotes apopotosis and mus-
due to diminished myotube formation (7, 9-11, 13, 30, cle atrophy by inhibiting E-protein/MRF dimeriza-
151, 152). Shefer et al. reported a reduced proliferation tion or by preventing MRFS binding to DNA (which
in aged myoblasts because there was only a 10 fold prevents muscle growth) (155-157).
increase in proliferation of myogenic cells from cul- As previously discussed, the muscle growth in-
tured aged (28 – 33 mo) mouse myofibers (at 6th day of hibitor myostatin may contribute to impaired muscle
plating relative to 4th day of plating), while myoblast repair (32, 73, 158, 159). Administration of recombi-
proliferation of young mouse (3 – 4 mo) myofiber nant myostatin to C2C12 cells (a skeletal muscle cell
cultures increased 15 fold at the same time point. Ad- line) inhibited myoblast differentiation and decreased
ditionally, there was a 24 h delay in myotube for- expression of MyoD, myf5, myogenin and p21 (159).
mation in the aged group, but no morphological dif- The authors suggested that myostatin may inhibit
ferences in myotube formation between the young MyoD through increased Smad 3 signaling (a media-
and aged groups were observed (9). Similarly, tor of myostatin signaling that inhibits myogenesis).
Conboy et al. reported fewer proliferating myoblasts MyoD expression was recovered when Smad 3 mol-
and myotubes from myofiber explants of aged (23 – 24 ecule was inhibited (through the expression of domi-
mo) relative to young (2 -3 mo) mouse muscle (7). nant-negative Smad 3 in C2C12 cells) (159). There may
These authors suggest that aged satellite cells retain be a positive-feedback mechanism in which myostatin
their myogenic potential but have an impaired ability may inhibit MRF expression, and the loss of MyoD

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Int. J. Biol. Sci. 2012, 8 740
and myogenin may further up-regulate myostatin that impaired satellite cell function in aged muscle
expression (73, 159). may contribute to the loss of myonuclei and
While there preponderance of evidence shows age-associated muscle atrophy (165).
decreased MRF and elevated myostatin in aged skel- Another intrinsic factor that contributes to ag-
etal muscle, it should be acknowledged that there are ing-associated impaired satellite cell function is alter-
reports stating either no change in MRF or myostatin ation of RNA pathways, including dysfunctional
expression, or elevations of MRF expression in aged pre-RNA processing within satellite cell nuclei that
muscle (6, 142, 160-162). Myogenin protein levels impacts gene expression (166). In addition, there are
were ~ 5.5-fold higher in gastrconemius of aged (32 alterations of ribonucleopreotein (RNP)-containing
mo) rats relative to young (4 mo) rats (142). Aged (31 structures within aged muscle (166, 167). In aged rat
mo) rats exposed to chemical injury displayed ele- satellite cell nuclei, there was accumulation of
vated myogenin mRNA levels at 21 days post-injury, rnRNPS (involved with pre-mRNA splicing), which
while myogenin mRNA returned to normal at that signified dysfunctional intranuclear transport of
time point in 3 mo old rats (6). Myostatin expression transcripts (166). Malatesta et al. also found decreased
in mouse gastrocnemius and plantaris increased from pre-mRNA transcription rate and decreased nucleo-
birth until 11 wks of age (161). Although there are plasmic splicing factors in myonuclei of aged myofi-
studies that suggest aging does not negatively affect bers (167). The authors suggest that mRNA processing
myogenic protein and myostatin expression, the au- is less efficient in aged than young muscle, and that
thors’ support the notion of decreased MRF in aged impaired mRNA function may contribute to the im-
skeletal muscle that affect muscle repair. The plethora paired satellite cell activity reported in aged muscle
of evidence offer viable mechanisms for impaired (166).
MRF expression which occur in aging tissue such as Cell fate choice is another intrinsic factor influ-
decreased protein synthesis, increased oxidative encing aged satellite cell function (14, 149). The in-
stress, as well as evidence of increased MRF inhibitor, creased presence of adipose tissue (such as adipocyte
myostatin. differentiation marker C/EBP) in aged muscle sug-
c. Cause of impaired aged skeletal muscle repair gests that satellite cells may prefer an altered cell fate.
– intrinsic factors: The intrinsic alterations within Instead of choosing myogenic cells, the satellite cells
satellite cells that occur during aging negatively affect commit to non-myogenic cell fates including adipo-
satellite cell integrity. With aging, satellite cells may genic or fibrogenic cells (149). There was greater ex-
have reached their “Hayflick limit” and reached their pression of adipogenic genes (fatty acid binding pro-
cell division threshold, which would result in de- tein aP2) in myoblasts cultured from aged (23 mo)
creased satellite number and ultimately impaired re- relative to young (8 mo) mouse hindlimb muscles
generation (8). In addition, there is increased apopto- (168). The change in cell fate decision away from
sis in aged satellite cells and myonuclei, which further myogenesis in aged skeletal muscle may be associated
depletes the satellite cell pool as judged by differences with decreased myogenic capacity and increased ad-
between aged (31 mo) and young (3 mo) rat EDL sat- ipogenesis (168). Aged satellite cells may also choose
ellite cells (33, 163). Cultured aged myofibers with fibrogenesis over myogenic cell fate (14). Aged myo-
depleted Pax7+ satellite cells became apoptotic re- fiber explant cultures had increased fibronectin ex-
sulting in few differentiated myotubes (164). pression relative to young myofiber explants
Cell size regulation is another intrinsic factor cultures(14). A satellite cell’s preference for adipo-
that may impact satellite cells (165). Brack et al. sug- genic or fibrogenic cell fate would result in an im-
gest that nuclear domain size is maintained with age pairment in the regenerative potential of aged skeletal
because the age-associated loss of myonuclei, includ- muscle.
ing loss of satellite cells, causes the cytoplasm to di- d. Cause of impaired aged skeletal muscle repair
minish and the size of the muscle fiber decreases. – microenvironment mileu: Another reason for the
Whether the loss of myofiber nuclei causes muscle delay in the aged myogenic program may be the
atrophy, or if muscle atrophy causes the loss of my- composition of the environmental mileu surrounding
onuclei is controversial. Brack et al. suggest that im- the satellite cells (9, 13, 139, 145, 169-172). Young
paired satellite cell function or quantity may contrib- muscle transplantation into aged tissue milieu results
ute to the age-associated loss of myonuclei. Although in myogenic delay (141, 170, 172). In addition, there is
there is controversy about the ability of aged muscle decreased satellite cell proliferation and expression of
to regulate nuclear domain size, Brack et al. propose myoblast markers desmin and myf5 in satellite cells
that effective satellite cell function may be critical for exposed to serum from aged muscle (170). Further-
maintaining nuclear domain and cell size control, and more, when young (2 mo) or aged (15 – 21 mo) mouse

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