Sarcopenia Age-Related Muscle Wasting and Weakness: Mechanisms and Treatments P30 potx

276 K. O’Connell et al.
The chaperone cvHsp appears to counter-act deleterious protein aggregation in
the cytosol, sarcolemma and actomyosin apparatus of aged muscle (Doran et al.
2007c). In addition, increased concentrations of the ubiquitous small heat shock
protein aB-crystallin were also detected by the proteomic profiling of senescent
fibres (Doran et al. 2008). The family of small heat shock proteins quickly
responds during stressful conditions and facilitates the disintegration of poly-
disperse assemblies into smaller subunits. This process is ATP-independent
whereby small chaperone subunits bind to unfolding substrate and then reform
into larger complexes (Stamler et al. 2005). The age-dependent activation of the
cytoprotective protein complement of skeletal muscles seems to counter-act
increased levels of denatured proteins in senescent fibres, especially abundant
elements such as non-functional myosins, actins, troponins and tropomysoins
(Vandervoort 2002; Prochniewicz et al. 2007). Increased chaperone levels repre-
sent an essential cellular rescue mechanism for eliminating the potentially
destructive accumulation of inactive muscle protein aggregates. During aging,
adaptive fibre transformation occurs in skeletal muscles. The fast-to-slow transi-
tion process encompasses major cellular remodeling. This includes the degenera-
tion of the fastest-twitching fibre population, the activation of the satellite pool of
muscle precursor cells and a certain degree of phenotypic fibre shifting within a
contractile unit. However, since senescent muscles have a reduced regenerative
capacity, adaptive fibre modulation probably triggers excessive detrimental pro-
tein aggregation as compared to healthy adult tissues. This in turn requires a
massive cellular stress response to prevent contractile dysfunction. Therefore, in
the context of a blunted stress response involving large heat shock proteins in
aged muscle (Kayani et al. 2008), the drastic up-regulation of low-molecular-
mass chaperones probably represents a compensatory mechanism that mostly
supports filament remodeling (Doran et al. 2007c).
Continuous contractile activity clearly influences the expression of heat shock
proteins (Neufer et al. 1998). Key chaperones containing the a-crystallin domain
are up-regulated following chronic contraction patterns (Donoghue et al. 2007). In

analogy to chronic neuromuscular activity, similar fibre transition processes occur
in aged muscle. The concomitant damage to the actomyosin apparatus and associ-
ated cytoskeletal network may therefore trigger an increased synthesis of small heat
shock proteins (Doran et al. 2009a). In contrast, cellular stress does not generate a
sufficient response by larger heat shock proteins, such as those encoded by the
Hsp70 gene (Liu et al. 2006). The up-regulation of Hsp70 and related chaperones
is usually part of a highly coordinated stress response that prevents extensive mus-
cular atrophy by limiting the stress-induced rate of cellular degeneration (Chung
and Ng 2006). High levels of Hsp70 are essential for the stabilization of metabolic
pathways, the prevention of high rates of apoptosis and the facilitation of physio-
logical adaptation to changed functional demands. An age-related impairment of
the Hsp70 response is believed to play a key role in contractile deficits (McArdle
et al. 2004). It is therefore not surprising that skeletal muscles of aged transgenic
mice with over-expressed levels of Hsp70 are partially protected against fibre
degeneration (Broome et al. 2006).
277Proteomic and Biochemical Profiling of Aged Skeletal Muscle
This suggests that a well-designed pharmacological approach to enhance the
natural stress response could potentially eliminate excessive fibre damage in aged
muscle. In other areas of biomedicine, the drug-induced modulation of the cellular
stress response has already gained considerable attention, as reviewed by Soti et al.
(2005). Various inducers, co-inducers and inhibitors of specific heat shock proteins
are currently evaluated as emerging therapeutic vehicles for the treatment of heart
disease, diabetes, cancer and neurodegenerative disorders (Calderwood et al. 2006;
Shamaei-Tousi et al. 2007). Since the up-regulation of small heat shock proteins,
such as aB-crystallin or cvHsp, may represent an auto-protective mechanism in
senescent muscle, a further increase in their expression levels may have therapeutic
benefits. Hence, a pharmacologically mediated increase in essential muscle chap-
erones may be a realistic treatment option for eliminating certain neuromuscular
impairments and could decisively improve the survival rate of stressed motor units
in the senescent body.

3.4 Excitation–Contraction Uncoupling in Aged Muscle
Ca
2+
-fluxes represent one of the most crucial second messenger system in contrac-
tile tissues (Berchtold et al. 2000). Alterations in Ca
2+
-levels do not only affect
protein activity and key physiological processes, but also gene expression patterns
in skeletal muscle. Changes in the cytosolic Ca
2+
-concentration play a key role in
myogenesis, differentiation, fibre transformation, metabolic regulation, excitation–
contraction coupling and muscle relaxation. Importantly, cyclic alterations in cyto-
solic Ca
2+
-levels determine the contractile status of skeletal muscle fibres. The
regulation of Ca
2+
-homoeostasis and the mediation of the excitation–contraction–
relaxation cycle depend on a finely tuned interplay between voltage-sensing recep-
tors in the transverse tubules, Ca
2+
-release channel units in the junctional
sarcoplasmic reticulum, luminal and cytosolic Ca
2+
-binding proteins, and Ca
2+
-
pumps of the sarcoplasmic reticulum, as well as minor structural components of the
triad junction and sarcolemmal ion-regulatory elements such as ion exchangers and

ion pumps (Murray et al. 1998). It is therefore not surprising that abnormal Ca
2+
-
handling is involved in a variety of muscle pathologies (MacLennan 2000;
Froemming and Ohlendieck 2001), including sarcopenia of old age (Renganathan
et al. 1997; O’Connell et al. 2008b).
The physical coupling between the voltage-sensing a
1S
-subunit of the trans-
verse-tubular dihydropyridine receptor and the ryanodine receptor Ca
2+
-release
channel of the junctional sarcoplasmic reticulum forms the central signal transduc-
tion unit during excitation–contraction coupling in mature skeletal muscles
(MacLennan et al. 2002). The dihydropyridine receptor from skeletal muscle con-
sists of a a
1S
-a
2
/d-b-g configuration. The a
1S
-subunit represents the principal ion
channel pore with three cytoplasmic loops between four repeat segments, whereby
the II-III loop domain interacts directly with the junctional calcium release channel.
During muscle aging, a drastically lowered supply of Ca
2+
-ions to contractile
278 K. O’Connell et al.
proteins occurs due to uncoupling between the two main triad receptors
(Renganathan et al. 1997). Excitation–contraction uncoupling appears to be due to

a larger number of ryanodine receptors being uncoupled to the voltage-sensing
dihydropyridine receptor units as compared to mature fibres. A pathophysiological
disconnection between sarcolemmal excitation and muscle contraction may result
in alterations in the voltage-gated Ca
2+
-release mechanism, decreases in myoplas-
mic Ca
2+
-elevation in response to surface depolarisation, reduced Ca
2+
-supply to the
actomyosin apparatus and reduced contractile strength. Thus, abnormal Ca
2+
-
handling may account for a significant proportion of the decay in skeletal muscle
force during aging (Delbono et al. 1995). A recent immunoblotting and immuno-
fluorescence survey has confirmed the excitation–contraction coupling hypothesis.
The Ca
2+
-binding protein named sarcalumenin, which represents a major mediator
of ion shuttling within the longitudinal sarcoplasmic reticulum, was shown to be
greatly reduced in aged rat gastrocnemius muscle as compared to young adult
specimens (O’Connell et al. 2008b). In addition, key elements of the plasmalemma-
associated Ca
2+
-extrusion system, i.e. the calmodulin-dependent Ca
2+
-ATPase and
the Na
+

-Ca
2+
-exchanger, were also found to be diminished in aged muscle.
Figure 4 summarizes the findings of the immunoblotting survey of essential physi-
ological regulators of Ca
2+
-homeostasis and how their dysregulation may affect the
excitation–contraction–relaxation cycle during aging. The overall protein band pat-
tern of electrophoretically separated crude tissue extracts from 3-month versus
30-month old rat gastrocnemius muscle was very comparable between young adult
versus senescent fibres. The previously reported senescence-related decrease in the
a
1S
-subunit of the dihydropryridine receptor, but not its auxiliary a
2
-subunit, was
confirmed. Immunoblotting of the sarcoplasmic reticulum proteins that mediate Ca
2+
-
buffering and Ca
2+
-removal, i.e. fast and slow calsequestrins and the Ca
2+
-pumping
ATPase isoforms SERCA1 and SERCA2, suggested a shift to a slower phenotype, but
these findings are not statistically significant. In contrast, the reduced expression of
the 160 kDa Ca
2+
-binding protein sarcalumenin and its related glycoprotein product
of 53 kDa, as well as the Na

+
-Ca
2+
-exchanger and the PMCA-type Ca
2+
-ATPase was
shown to be significant in aged muscle. Thus, downstream from the coupling defect
between the dihydropyridine receptor and the junctional Ca
2+
-release channel, addi-
tional age-dependent changes appear to exist in Ca
2+
-regulatory elements. Reduced
levels of sarcalumenin and the two sarcolemmal Ca
2+
-extrusion proteins may cause
abnormal luminal Ca
2+
-binding and impaired Ca
2+
-removal (O’Connell et al. 2008b).
This in turn could exacerbate disturbed ion fluxes and diminished triad signaling in
senescent muscle and thereby contribute to contractile weakness.
4 Conclusion
Natural aging is a fundamental biological process. The functional decline of
skeletal muscle fibres and the loss of total muscle mass are crucial factors that
render the human body more susceptible to a metabolic disequilibrium and physical
279Proteomic and Biochemical Profiling of Aged Skeletal Muscle
weakness. Besides studying the histological and anatomical effects of muscle aging
on frailty and fragility, it is also crucial to determine the molecular mechanisms that

underlie age-dependent alterations at the cellular level. The application of modern
proteomic methodology for analysing age-related impairments in contractile tissues
promises to elucidate the pathobiochemical processes that lead to sarcopenia of old
age. Mass spectrometry represents an unrivalled technique for the swift and reliable
identification of protein factors involved in pathological pathways or compensatory
Fig. 4 Overview of the excitation–contraction uncoupling hypothesis of skeletal muscle aging
and comparative immunoblot analysis of key Ca
2+
-handling proteins in young adult versus
senescent muscle. Shown is a Coomassie-stained gel and immunoblots of young adult versus aged
rat gastrocnemius preparations. Immunoblots were labeled with antibodies to key proteins of the
sarcolemma (SL), transverse tubules (TT) and sarcoplasmic reticulum (SR), including sarcalumenin
(SAR) and its alternative splice product, the 53 kDa sarcoplasmic reticulum glycoprotein (53-
SRGP), fast and slow calsequsetrin (fast CSQf; slow CSQs), fast and slow sarcoplasmic reticulum
Ca
2+
-ATPase (fast SERCA1; slow SERCA2), the Na
+
-Ca
2+
-exchanger (NCX), the plasmalemmal
Ca
2+
-ATPase (PMCA), and the a
1S
- and a
2
-subunit of the dihydropryridine receptor (DHPR).
Molecular mass standards (in kDa) are indicated on the left of the Coomassie-stained gel panel.
The comparative blotting was statistically evaluated using an unpaired Student’s t-test (n = 6;

*p < 0.05; **p < 0.01. Standard methods were employed for muscle preparations from crude
tissue extracts, one-dimensional gel electrophoresis and immunoblot analysis of Ca
2+
-handling
proteins (O’Connell et al. 2007). The central panel outlines the dysregulation of Ca
2+
-fluxes in
senescent fibres and how this may affect the excitation–contraction–relaxation cycle during
skeletal muscle aging. Besides the Ca
2+
-handling proteins that have been analysed by
immunoblotting, other key elements of ion homeostasis and muscle regulation are included in this
diagram, i.e. the ryanodine receptor (RyR) Ca
2+
-release channel of the sarcoplasmic reticulum and
the troponin subunit TnC
280 K. O’Connell et al.
mechanisms involved in aging. Over the last few years, mass spectrometry-based
proteomics has identified a large number of relatively sarcopenia-specific biomark-
ers. Skeletal muscle proteins that exhibit altered expression levels or changed post-
translational modifications during aging include regulatory proteins, contractile
elements, metabolic enzymes and cellular stress proteins. The complexity of the
observed changes in the senescent muscle proteome confirm the idea that sarcope-
nia is probably based on a multi-factorial etiology, rather than alterations in just one
class of protein factors, regulatory mechanisms or aging-inducing gene clusters.
Proteomic profiling studies have established distinct switches in fibre type-specific
isoforms of contractile and metabolic proteins during aging, demonstrating an age-
related transformation to slower-twitching muscles. The fast-to-slow transition
process is accompanied by bioenergetic adaptation mechanisms. The comparative
proteomic analysis of adult versus senescent muscles has clearly revealed a drastic

shift to more aerobic-oxidative metabolism during aging. The proteomic identifica-
tion of new sarcopenic biomarkers and their detailed cell biological, physiological
and biochemical characterzation will hopefully lead to the prompt development of
superior diagnostic tools and the improved design of pharmacological strategies to
counter-act the age-induced loss of contractile tissue. Since alterations in the neu-
romuscular system are of central importance for comprehending the overall patho-
genesis of the aging process in humans, the recent findings from proteomic studies
will be crucial for improving our general biomedical knowledge on the mechanisms
of aging.
Acknowledgements Research in the author’s laboratory was supported by a principal investiga-
tor grant from Science Foundation Ireland (SFI-04/IN3/B614) and equipment grants from the Irish
Health Research Board (HRB-EQ/2003/3, HRB-EQ/2004/2) and the Higher Education Authority
(HEA-RERGS-07-NUIM). The authors thank Dr. Marina Lynch (Trinity College Dublin) for her
generous help obtaining aged rat muscle, and Ms. Caroline Batchlor (NUI Maynooth) for assis-
tance with mass spectrometry.
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