BACKGROUND
ageing affects most tissues and physiologic functions, and one of the most affected organs is the skeletal muscle.
the progressive decline in muscle mass and function due to ageing, which is also referred to as sarcopenia  <cit> , contributes to both loss of autonomy  <cit> , increased prevalence of falls, decreased resistance to metabolic aggression that increases morbidity  <cit>  and mortality  <cit> .
numerous theories have been proposed to explain muscle ageing.
obviously, this is a multifactorial phenomenon which implicates intrinsic factors such as perturbations in the endocrine system, neuronal remodelling, oxidative stress and deficiencies in muscle regeneration, extrinsic factors such as diet and exercise, and also probably other unknown mechanisms  <cit> .
age-related degenerative changes are reflected in alterations in muscle morphology, function, and biochemical properties.
muscle ageing is thus associated with muscle fiber atrophy  <cit> , reduced muscle regenerative capacity  <cit> , and neuropathic processes leading to motor unit denervation  <cit> .
mitochondrial dysfunctions with decreased capacity of oxidative enzymes and a decline of mitochondrial atp production may also be observed with ageing in skeletal muscles .
the overall functional, structural, and biochemical alterations in ageing muscle have been extensively studied, but the molecular mechanisms implicated remain to be specified.
the differential expression profiles of mrnas constitute a first essential level of information, but analyses of the expression profile of proteins in ageing are also required to understand the molecular mechanisms important for the muscle ageing process  <cit> .
in fact, unlike the genome, the proteome varies in response to many physiological or pathological factors.
in addition, the proteome is orders of magnitude more complex than the transcriptome due to post-translational modifications, protein oxidation or limited protein degradation  <cit> .
several studies have been conducted in rat muscle and proteomic profiling has demonstrated substantial alterations in muscle proteins involved in key metabolic pathways, myofibrillar remodelling, cytoskeleton organisation and mechanisms of cytoprotection and cytodetoxification .
however, few studies have been conducted with human muscle and results are contradictory.
gelfi et al.
<cit>  have shown that several enzymes involved in oxidative metabolism, including ubiquinol-cytochrome c reductase or aspartate aminotransferase, were more abundant in elderly than in young people, while short et al.
<cit>  have demonstrated a decrease in these enzymes with ageing.
in contrast, some results observed in rat muscle proteomic analyses were confirmed in human muscle, such as a decrease in enzymes involved in glycolytic metabolism and an increase in proteins involved in cytoprotection  and cytodetoxification   <cit> .
because epidemiological studies have indicated accelerated muscle wasting after the fifth decade with an approximately 2% reduction in muscle mass per year  <cit> , we have undertaken a top-down differential proteomic approach to determine potential changes after the fifth decade of life and to identify novel biomarkers of muscle ageing.
in a previous study, we used a shot-gun proteomics approach to identify  <dig> potential biomarkers  <cit> .
herein, we performed two dimensional gel electrophoreses  using biopsies of vastus lateralis from mature adult  vs. old  women, and for better separation of proteins, two different strategies were conducted.
firstly, overlapping immobilized ph gradient  with three different ph ranges were used to improve muscle proteins separation, and secondly, we assessed low salt extracts to remove the high abundance myofibrillar proteins and improve the detection of low abundance proteins.
the proteomic profiling of aged skeletal muscle fibers revealed a differential expression pattern of  <dig> potential biomarkers important for energy metabolism, contractile properties, calcium signaling, cytoprotection, regulation of protein misfolding, and proteolysis.
the present study demonstrates that alterations of muscle function in elderly women are associated with severe perturbed protein expression patterns and identified  <dig> new potential biomarkers of sarcopenia that had not previously been described.
RESULTS
differentially expressed proteins during ageing
in order to evaluate age-dependent alterations in the skeletal muscle proteome after the fifth decade of life, total muscle extracts from biopsies of mature adult  versus old  post-menopausal women were resolved by 2dge.
gels with overlapping range of ipgs were used to improve the separation of total muscle extracts.
2dge with medium range ipgs  revealed  <dig> protein spots that were matched between all individuals.
among these protein spots,  <dig> were found to be differentially expressed between adult and old women, and  <dig> were identified by liquid chromatography coupled to tandem mass spectrometry , corresponding to  <dig> different proteins .
2dge with acidic ipgs  distinguished  <dig> matched protein spots, and among them  <dig> were differentially expressed and identified as  <dig> different proteins .
narrow ipgs were also used to achieve optimal resolution in the ph  <dig> - <dig>  range, and this revealed a further  <dig> matched spots.
among these  <dig> spots, statistical analysis revealed  <dig> differentially expressed spots which were identified by lc-ms/ms as  <dig> different proteins .figure  <dig>
representative 2dge image obtained from total protein extracts of human
vastus lateralis
skeletal muscle.
2dge was performed using a ph range of 5– <dig> , 3– <dig>   or  <dig> - <dig>   in first dimension and sds-page  in the second.
protein loading was  <dig> μg, and the gels were stained using colloidal coomassie blue g- <dig>  differentially expressed and identified proteins are marked and spot numbers refer to table  <dig>
because myofibrillar proteins may hamper the detection of low abundance proteins, we also precipitated myofibrils at low ionic strength  <cit> , and focused on the soluble low ionic strength  extract.
thirty-six gels with medium range ipgs  were used to analyze lis extracts and  <dig> protein spots were matched between all individuals.
statistical analysis revealed that  <dig> spots were differentially expressed between adult and old women.
among them,  <dig> were identified by lc-ms/ms , and they corresponded to  <dig> different proteins.
targeting the lis sub-proteome improved the 2dge analysis of the muscle proteome, as most  differentially expressed lis proteins were not found in total muscle extracts.figure  <dig>
representative 2dge image obtained from low ionic strength  extracts of human
vastus lateralis
skeletal muscle.
2dge was performed using a ph range of 5– <dig> in the first dimension and sds-page  in the second.
protein loading was  <dig> μg, and the gels were stained using colloidal coomassie blue g- <dig>  differentially expressed and identified proteins are marked and spot numbers refer to table  <dig>
interestingly, shot-gun proteomics previously identified  <dig> potential biomarkers in the lis sub-proteome  <cit> .
it should be noted that, although shot-gun and 2dge proteomics are based on different strategies , the two techniques are complementary, as only  <dig> common proteins were found in both investigations: titin , ankyrin repeat domain-containing protein  <dig> , l-lactate dehydrogenase β , furamate hydratase , fatty acid binding protein  <dig>  and carbonic anhydrase  <dig> .
overall in the present 2dge study,  <dig> protein spots were matched between all individuals,  <dig> were differentially expressed and identified, and they corresponded to  <dig> different proteins.
table  <dig>  and table  <dig>  summarize the main properties of the proteins differentially regulated in skeletal muscle between adult and old post-menopausal women.
absolute fold changes varied from  <dig>  to  <dig> .
even though some fold-changes appeared relatively small, there were parallel changes of several components in each pathway or biological process, suggesting some important coordinated and specific regulations.table  <dig>
differentially expressed muscle proteins in total extract between adult  and old  post-menauposal women
perturbations of the myofilament network and cytoskeleton with ageing
sarcomeric proteins
muscle contraction is generated by an interaction between the molecular motor myosin and filamentous actin.
myosin is a hexameric protein that consists of two heavy chain subunits, two alkali light chain subunits and two regulatory light chain subunits.
myosin light chains typically exhibit various isoforms, and in our study, muscle ageing was associated with higher level of one myosin light chain 1/ <dig> skeletal muscle isoform  and lower level of one myosin regulatory light chain  <dig> ventricular/cardiac muscle isoform .
perturbations in myofibrillar contractile proteins were confirmed by the up-regulation of one isoform of fast troponin t  which is a major regulator of the thin filament.
troponin t directly interacts with key components in the thin filament regulatory system to mediate the activation and force development of actomyosin contractile units  <cit> .
age-related changes in fibers expressing various myosin light chain isoforms were previously described for myl <dig>  myl <dig> and tnnt <dig>  <cit> ; however, these changes were manifested differentially in distinct muscles.
interestingly, the old skeletal muscle also exhibited altered levels of several fragments of sarcomeric proteins.
thus, there were higher levels of n-terminal  and c-terminal  fragments of skeletal α-actin , and a higher level of a c-terminal fragment  of myosin- <dig> , the type iix adult fast myosin heavy chain.
the old muscle also exhibited lower level of a c-terminal fragment of ttn , when compared to adult muscle.
such fragments were not observed for non-sarcomeric proteins, and may indicate perturbations in sarcomeric proteolytic pathways.
a feature of human muscle ageing was also the increased expression of ankyrin repeat domain-containing protein  <dig> .
ankrd <dig> is a member of the mechano-sensing proteins that link myofibrillar stress response to muscle gene expression  <cit> .
ankrd <dig> interacts both with i-band sarcomeric proteins and with nuclear transcription factors  <cit> .
this stretch-response protein is preferentially expressed in slow type-i fibers, and is induced by denervation, which is consistent with neuronal remodeling in ageing muscle  <cit> .
aging of skeletal muscle involves a decrease in both total number  and size  of muscle fibers.
no consensus has been reached in the literature on whether hypoplasia is associated with a shift in fiber type distribution towards higher , lower  <cit>  or unaltered  <cit>  percentages of type-i versus type-ii fibers.
however, previous studies based on myosin heavy chain  histochemistry agreed that atrophy mostly affects type-ii fibers  <cit> .
in our 2dge analyses, there was no clear evidence for a fast-to-slow transition on the unique basis of the differential expression of myosin light chain and troponin isoforms.
cytoskeletal proteins
the integrity of muscle fibers depends on cytoskeletal components, which align sarcomeres and anchor them across the sarcolemma to the basement membrane  <cit> .
two isoforms of vinculin  were enhanced during ageing.
vcl localizes to adhesion junctions and is a central component of muscle costameres  <cit> .
vcl is placed between the integrin-talin complex and the actin cytoskeleton and is a major candidate for transduction of force during the contractile cycle.
besides binding to actin, vcl interacts with signaling networks and is important for signal transduction between the extracellular matrix and the cytoskeleton  <cit> .
finally, the four and a half lim domain protein  <dig> , which is down-regulated in our analysis, is an adaptor protein with numerous interaction partners.
in adult fibers, fhl <dig> interacts with cytoskeletal actin  <cit>  and co-localizes with integrin receptors at the periphery of z-discs  <cit> .
by binding to integrin and actin, fhl <dig> might then directly link the cytoskeleton to the extracellular matrix.
fhl <dig> also plays a role in myogenic progenitor cells , where it localizes to the nucleus and has been implicated in the regulation of proliferation  <cit>  and differentiation  <cit> .
vcl and fhl <dig> have not been previously identified by proteomic surveys of human skeletal muscle ageing  <cit> .
shot-gun proteomics confirmed the up-regulation of ankrd <dig>  <cit> , and western blotting experiments performed with total muscle extracts confirmed the differential expressions that we observed for vcl and fhl <dig> in lis extracts .
in all, the modifications that we report for sarcomeric actomyosin and cytoskeletal proteins are most likely related to disorganization of myofibers in old muscles.
moreover, these changes suggest an influence of the ageing process on the maintenance of the proper organization of sarcolemma in regular structures that are closely linked to costameres, and on the response to extracellular signals.
thus, the age-related changes in sarcomeric and cytoskeletal proteins may result in alterations in contractile properties and contribute to the development of sarcopenia.figure  <dig>
examples of differential expression of cytoskeletal proteins.
representative sections of 2dge images  and representative western blots  for vinculin   and four and a half lim domains  <dig>  .
in each panel, histograms represent normalized volume of protein spot , and western blot quantification  for adult and old post-menopausal women.
results are indicated as means ± se.
*: p < <dig>  indicates significant difference between adult and old women.
age-related changes in skeletal muscle affect signal transduction
in skeletal muscle, intracellular calcium  is an important secondary messenger for signal transduction and is essential for cellular processes such as excitation-contraction coupling.
action potentials elicit contractions by releasing ca2+ from the sarcoplasmic reticulum  via the ryanodine receptors .
ryrs are modulated directly or indirectly by various ions, small molecules and proteins, including calsequestrin.
in this study, proteomics analysis of muscle ageing identified an up-regulation of calsequestrin- <dig> , which is a major intra-sr ca2+ buffer that regulates the activity of ryrs  <cit> .
ryr receptors have several potential phosphorylation sites in their cytoplasmic domains and protein kinase a  has been shown to phosphorylate ryrs.
our analysis shows an increased level of the pka type iα regulatory subunit  during ageing.
among other substrates pka can phosphorylate ryr, and pka-mediated phosphorylation of ryr may result in leaky ryr channels and impaired ca2+ homeostasis  <cit> .
after initiation of muscle contraction by increasing cytoplasmic ca2+, ca2+ is pumped back to the sr by sarcoplasmic reticulum ca2+ atpase  leading to relaxation.
sarcalumenins  are major luminal glycoproteins that codistribute with serca and play a role in ca2+ transport and sequestration  <cit> .
there are two srl isoforms  that are generated by alternative splicing  <cit> .
in the present study, the 53-kda isoform  was found to be reduced in aged human muscle, and this reduction was confirmed by western-blotting .
the age-related decrease of srl is in agreement with previous studies in rat muscle indicating a shorter half-life of the 53-kda isoform  <cit>  and a lower level of the 160-kda isoform  <cit> .figure  <dig>
examples of differential expression of proteins implicated in signal transduction.
representative sections of 2dge images  and representative western blots  for sarcalumenin  , myozenin- <dig>  , annexin a <dig>  , annexin a <dig>   and glutathione s-transferase omega- <dig>  .
in each panel, histograms represent normalized volume of protein spot , and western blot quantification  for adult and old post-menopausal women.
results are indicated as means ± se.
*: p < <dig>  indicates significant difference between adult and old women.
we have also identified an up-regulation of myozenin- <dig>  which is expressed in fast-twitch fibers of skeletal muscle and modulates the function and substrate specificity of calcineurin, a ca2+/calmodulin-dependent serine-threonine phosphatase that plays an important role in transducing calcium-dependent signals  <cit> .
this increase in myoz <dig> was also confirmed by western-blotting .
in skeletal muscle, numerous proteins can bind ca2+, and muscle ageing was further associated with higher level of three members of the annexin family which undergo ca2+-dependent binding to the cellular membranes.
we identified annexin a <dig> , a <dig>  and a <dig> .
annexins have been involved in a broad range of molecular and cellular processes.
noteworthy anxa <dig> may contribute to the regeneration of skeletal muscle tissue by modulating migration  <cit>  and fusion  <cit>  of satellite cells.
anxa <dig> is overexpressed in different muscular dystrophies  <cit>  and can further participate in sarcolemmal and t-tubular repair processes  <cit> .
anxa <dig> also promotes membrane repair by self-assembling into two-dimensional arrays on membranes  <cit> .
anxa <dig> was originally described as a protein that provokes fusion of lipid vesicules  <cit> .
more recently, anxa <dig> was identified as an essential protein for autophagy induction by modulating the intracellular ca2+ concentration  <cit> .
western-blotting experiments confirmed the overexpression of anxa <dig> and anxa <dig>  in total extracts of old muscle, compared to adult muscle.
glutathione s-transferase omega- <dig>  is distinguished from the other glutathione s-transferase family members by a different active center amino acid residue, which results in loss of prototypical glutathione conjugating activity  <cit> .
instead, human gsto <dig> is reported to potentiate skeletal muscle ryanodine receptor   <cit> .
the age-related down-regulation of gsto <dig>  was confirmed by western-blot , and may thereby be involved in the impairment of ca2+ homeostasis.
most of these signal transduction proteins  have never been reported in previous proteomic studies of muscle ageing.
overall, our findings suggest significant alterations in ca2+ signaling which may be important for the age-related modifications in muscle contractile properties and may contribute to muscle weakness.
perturbations in the energy metabolism of old muscle
disturbance in energy metabolism is another characteristic feature of old muscles.
decreased activities of glycolytic enzymes were previously reported in rat  <cit>  and in human  <cit>  skeletal muscles, whereas expression of mitochondrial enzymes was more controversial in the literature  <cit> .
our study points to an age-associated decline in key enzymes of the glycolytic, krebs cycle and oxidative phosphorylation pathways.
cytoplasmic energy metabolism
in the current study, perturbations in the energy metabolism of old muscle were indicated by the down-regulation of five isoforms of monomeric creatine kinase  .
sds-stable dimeric and trimeric forms of ckm were previously described in mice muscle  <cit> , and we also observed age-related decreases in dimeric  and trimeric  forms of ckm.
creatine/phosphocreatine is central to maintain energetic homeostasis as it connects intracellular sites of energy demand with sites of atp production.
at these sites, ckm catalyses the transphosphorylation between phosphocreatine and adp.
in sarcomeric m-line, ckm interacts with myomesin and supplies atp for the actomyosin contractile unit.
the first step of glycogenolysis pathway is the production of glucose-6-phosphate by glycogen phosphorylase , which catalyzes the phosphorolytic cleavage of a glucosyl residue from the glycogen polymer.
four isoforms  of pygm increased, and one  decreased with ageing.
the resulting glucose 1-phosphate molecule is converted by phosphoglucomutase to glucose 6-phosphate.
proteomic analysis revealed a decreased expression of phosphoglucomutase- <dig>  in elderly women.
several glycolytic enzymes were also down-regulated with ageing.
enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate.
in the muscle of post-menopausal women, four isoforms of the muscle specific β-enolase  were selectively decreased during ageing.
we also detected lower levels of ldhb  which catalyzes the inter-conversion of pyruvate  and lactate with concomitant inter-conversion of nadh and nad+.
finally, down-regulations of glycerol-3-phosphate dehydrogenase  and glycerol-3-phosphate dehydrogenase 1-like protein  were observed.
cytosolic gpd <dig>  together with its mitochondrial isoform, constitute the gpd <dig> shuttle, which is essential for mitochondrial oxidation of glycolytic nadh.
although less active than its gpd <dig> counterpart, gpd1l exhibits dehydrogenase activity, and is also implicated in the regulation of hypoxia  <cit> .
age-related decline in cytoplasmic glycerol-3-phosphate dehydrogenase may indicate reduced mitochondrial oxidation of cytosolic nadh in old muscle.
triosephosphate isomerase , that catalyzes the isomerization of the dihydroxyacetone phosphate  and d-glyceraldehyde 3-phosphate, was the only glycolytic enzyme more abundant in old muscle.
tpi <dig> ensures that dhap produced by aldolase is further metabolized by the glycolytic enzymes.
impairment of tpi <dig> can result in chemical conversion of dhap into toxic methylglyoxal  <cit>  promoting the formation of advanced glycation end-products.
in the old muscle, an increased level of tpi <dig> may then represent a compensatory adaptation to avoid excessive formation of toxic products  <cit> .
mitochondrial energy metabolism
alterations in the mitochondrial krebs cycle were revealed by the age-dependent reduction in two subunits of the pyruvate dehydrogenase, pyruvate dehydrogenase e <dig> component subunit β  and dihydrolipoyl dehydrogenase , and by the reduction in aconitate hydratase  and fh , two enzymes that catalyze the isomerization of citrate to isocitrate and the hydration of furamate to malate, respectively.
moreover, alterations in oxidative phosphorylation were also revealed by lower levels of three components of the respiratory chain, nadh dehydrogenase iron-sulfur protein  <dig> of complex i , cytochrome b-c <dig> complex subunit rieske of complex iii  and subunit 5a of cytochrome c oxidase of complex iv .
ageing was further associated with lower level of atp synthase subunit β .
among these proteins implicated in energy metabolism, two  have never been reported in previous muscle ageing studies, and fh and atp5b were similarly found in our shot-gun experiment  <cit> .
western blotting experiments confirmed alterations in glycolytic and oxidative metabolism, as we observed decreased expression of eno <dig>  gpd <dig>  ndufs <dig> and uqcrfs <dig>  in total extracts of old muscle compared to adult muscle .
concomitant decreases of key enzymes of krebs cycle and major complexes of oxidative phosphorylation provided evidences for alteration of mitochondrial metabolism in the old skeletal muscle.figure  <dig>
examples of differential expression of proteins implicated in energy metabolism.
representative sections of 2dge images  and representative western blots  for β-enolase  , glycerol-3-phosphate dehydrogenase   , nadh dehydrogenase  fe-s protein  <dig>   and cytochrome b-c <dig> complex subunit rieske  .
in each panel, histograms represent normalized volume of protein spot , and western blot quantification  for adult and old post-menopausal women.
results are indicated as means ± se.
*: p < <dig>  indicates significant difference between adult and old women.
lipid metabolism
in addition to glucose, lipids are another source of energy in skeletal muscle.
several intracellular fatty acid-binding proteins  have been identified.
they have important functions in the transport of intracellular fatty acids by increasing their solubility and have been shown to enhance the transport of fatty acids from the cell membrane to the site of oxidation, i.e.
the mitochondria, and to the site of esterification into intramyocellular triglycerol  <cit> .
muscle ageing was associated with the down-regulation of heart fabp , the major fabp in skeletal muscle, and the up-regulation of adipocyte fabp .
fabp <dig> plays an important, but merely permissive role in fatty acid uptake by skeletal muscles  <cit> .
fabp <dig> is an adipocyte marker, but is also expressed in muscle fibers  <cit> .
therefore, the age-related up-regulation of fabp <dig> may relate to increased number of adipocytes, and/or to an increased expression in muscle fibers.
except in our shot-gun study  <cit> , fabp <dig> was not noticed in previous reports on sarcopenia.
of note, the lipid chaperone fabp <dig>  was also identified as a predominant positive regulator of toxic lipid-induced endoplasmic reticulum  stress  <cit> , and fabp <dig> up-regulation could suggest increased er stress in old muscle.
overall, our proteomic analyses therefore provided strong evidences for a decline in both glycolytic and mitochondrial energy metabolism in the old skeletal muscle.
declines in mitochondrial oxidative capacity were previously reported with advancing age  <cit> , although physical activity, rather than chronological age, was also reported to be the primary determinant  <cit> .
because the activity score was significantly higher for the adult than for the old women group, future studies will be required to specify whether mitochondrial oxidative capacity still decreases in old subjects maintaining physical activity scores.
detoxification of cytotoxic products and cytoprotection in the old muscle
protection against mitochondrial oxidative stress
mitochondrial dysfunctions may lead to an excessive production of reactive oxygen species , and accumulating evidences suggest that oxidative stress underlies the ageing process in skeletal muscle  <cit> .
the removal of h2o <dig> in cells is mediated by catalase , glutathione peroxidase and peroxiredoxin   <cit> .
in the present study, ageing of human skeletal muscle was associated with lower levels of cat  and higher levels of prdx <dig> .
because cat is localized preferentially in peroxisomes  <cit> , its function is limited to the inactivation of h2o <dig> diffusing into these organelles.
in contrast, prdx <dig> is the only peroxiredoxin restricted to mitochondria, and high levels of prdx <dig> may provide a primary line of defence against h2o <dig> over-produced by the respiratory chain in old muscle mitochondria.
neither cat nor prdx <dig> have previously been reported in proteomic analyses of muscle ageing.
a wide range of activities have been reported for protein dj- <dig> , however, there is consensus that park <dig> is responsive and protective against mitochondrial oxidative stress  <cit> .
park <dig> is an atypical peroxiredoxin-like peroxidase  <cit> .
during oxidative attack, park <dig> is relocalized to mitochondria, has a functional role in scavenging mitochondrial h2o <dig> and decreases mitochondrial fragmentation  <cit> .
therefore elevated levels of park <dig> in the old muscle may be important in regulating cellular antioxidant capacity.
detoxification of cytotoxic products
oxidative stress increases the production of cytotoxic aldehydes, which can react with cellular proteins, nucleic acids and cell membranes.
protection against reactive aldehydes is provided by several families of detoxification enzymes, including aldo-keto reductase  and aldehyde dehydrogenase .
our proteomic analysis provided evidences for perturbed scavenging of reactive aldehyde products in the old muscle, as aldehyde dehydrogenase  and alcohol dehydrogenase , were up-regulated, while aldose reductase  and delta-1-pyrroline-5-carboxylate dehydrogenase  decreased with ageing.
similar regulations were previously reported during ageing in rat skeletal muscle for aldh <dig>  <cit>  and akr1b <dig>  <cit> .
however, no previous study of muscle ageing has identified the differential expression of akr1a <dig> and aldh4a <dig>
the mitochondrial enzyme aldh <dig> detoxifies aromatic and aliphatic aldehydes , which are produced during oxidative stress as a result of lipid peroxidation  <cit> .
the cytosolic oxidoreductase akr1a <dig> has broad substrate specificity, and similarly catalyzes the reduction of aliphatic and aromatic aldehydes, ketones, and xenobiotics  <cit> .
the up-regulations of aldh <dig> and akr1a <dig> thus suggest enhanced scavenging of reactive aldehyde products in the old skeletal muscle.
our western blot analyses confirmed that muscle ageing is associated with higher levels of aldh <dig> .figure  <dig>
examples of differential expression of proteins implicated in cytoprotection, cytodetoxification or proteolysis.
representative sections of 2dge images  and representative western blots  for aldehyde dehydrogenase  , α-crystallin b chain  , heat shock  <dig> kda protein 1a/1b  , hsp 90-beta  , and transitional endoplasmic reticulum atpase  .
in each panel, histograms represent normalized volume of protein spot , and western blot quantification  for adult and old post-menopausal women.
results are indicated as means ± se.
*: p < <dig>  indicates significant difference between adult and old women.
while aldh <dig> and akr1a <dig> act principally as detoxification enzymes, akr1b <dig> and aldh4a <dig> have additional roles besides that of detoxification.
accumulating evidence attributes a significant role to akr1b <dig> in transducing cytotoxic signals initiated by inflammatory cytokines  <cit> .
akr1b <dig> inhibitors have been reported to disrupt signalling cascades leading to nfkb activation  <cit> , and reduced levels of akr1b <dig> may therefore be important to limit activation of the nfkb pathway in the old muscle.
aldh4a <dig> amino acid sequence diverges from the other aldh  <cit>  and this enzyme is mostly involved in metabolic regulation  <cit> .
aldh4a <dig> is metabolically important, since its substrate  appears as a common intermediate in the degradative and biosynthetic pathways of the amino acids arginine, citrulline, ornithine and proline to and from glutamic acid.
the down regulation of aldh4a <dig> may thus indicate reduced intermediary metabolism in the old skeletal muscle.
quality control of cellular proteins
to detect, refold, and eventually eliminate abnormal proteins, cells use quality control mechanisms that buffer protein homeostasis  against cellular stress.
the proteomic analysis described here demonstrated the differential regulation of  <dig> spots identified as heat shock proteins .
all were increased with ageing and they encompassed  <dig> hsp groups in human: hspa, hspb and hspc  <cit> .
the small heat shock proteins  are atp-independent chaperones.
hspbs prevent the aggregation of improperly folded or partially denatured proteins, and are involved in their transfer to the atp-dependent chaperones or to the protein degradation processes such as proteasomes or autophagosomes  <cit> .
three hspbs displayed higher levels in old women compared to adult women: heat shock protein beta- <dig> , alpha-crystallin b chain  and heat shock protein beta- <dig> .
similar up-regulation of hspb <dig>  hspb <dig> and hspb <dig> have been previously reported for rat muscle ageing  <cit> .
our western blot experiments performed with total muscle extracts confirmed that the old human muscle exhibited higher levels of hspb <dig> than adult muscle .
in addition to chaperone activities, hspb <dig> and hspb <dig> have the ability to control the redox status, protect the actin cytoskeleton  <cit>  and inhibit apoptotic cell death  <cit> .
the atp-dependent chaperones of the hspa group  are essential for proteostasis as they contribute to the folding and assembly of nascent polypeptides, the transport of proteins across membranes, and the selection of misfolded proteins for degradation  <cit> .
muscle ageing was associated with the up-regulation of two hspa proteins: the heat shock  <dig> kda protein 1a/1b , and the mitochondrial stress- <dig> protein .
none of these hspa have been previously reported in proteomic analyses of muscle ageing, and our western blot experiments confirmed the age-related up-regulation of hspa1a .
hspa1a is the most abundant inducible cytoprotective hsp <dig> chaperone.
notably in rodents, overexpression of hspa1a has been repeatedly reported to attenuate muscle atrophy induced by immobilization  <cit> , lengthening contractions  <cit>  or cryo-lesions  <cit> .
it is therefore likely that the old human muscle may increase hspa1a levels as a protection against muscle atrophy.
hspa <dig> is a central component of the mitochondrial protein import motor and it plays a key role in the folding of matrix-localized mitochondrial proteins.
hspa <dig> is the only known mitochondrial hsp <dig> chaperone, and thus serves as a unique scavenger of toxic protein aggregates in human mitochondria  <cit> .
one hspc was further found to be up-regulated in these ageing muscles: the heat shock protein hsp  <dig> .
hsp  <dig>  which has not been identified in previous muscle ageing studies, is another important chaperone that functions downstream of hspa in the atp-dependent folding and conformational regulation of many client proteins, including protein kinases, steroid receptors, endothelial no synthase and transcription factors  <cit> .
most of the clients, with which hsp  <dig> interacts, are thus involved in signal transduction, making hsp  <dig> a critical factor in cell signalling.
western blot experiments confirmed the age-related up-regulation of hsp 90-beta .
finally and in addition to the hsp proteins, the chaperone operating in the endoplasmic reticulum , protein disulfide-isomerase a <dig> , was also found to increase with human muscle ageing, and this observation is in agreement with previous studies in rat  <cit> .
pdia <dig> is a multifunctional protein with thiol-protein disulphide oxidoreductase activity, which ensures proper folding of glycoproteins and assembly of major histocompatibility complex class i complex  <cit> .
outside the er, pdia <dig> also functions as a plasma membrane receptor for 1α,25-dihydroxy vitamin d <dig>  <cit> , which might be important with regard to the implication of vitamin d in ageing  <cit> .
cytoprotection in the old skeletal muscle
in agreement with a previous study in rat  <cit> , higher levels of two isoforms of carbonic anhydrase  <dig>  were identified in senescent human muscle.
no consensus has been reached in the literature about ca <dig>  as both increased  <cit>  and decreased levels  <cit>  were observed in old muscles.
our analyses of muscle ageing in women indicated an overexpression of  <dig> isoforms of ca <dig>  and this was confirmed by our shot-gun study  <cit> .
the various isoforms of cas play a crucial role in co2-removal and co2-provision for metabolic processes, and are central for the acid–base balance and the regulatory processes of ion homeostasis.
numerous proteins are involved in ion homeostasis, including the selenium-binding protein  <dig>  of which two isoforms were increased  and one isoform was decreased  in ageing.
proteolytic systems
muscle proteins are continuously turning over, and cells contain multiple proteolytic systems to carry out the degradation process.
several components of the major proteolytic systems, i.e.
ubiquitin-proteasome  <cit>  and lysosomal autophagy  <cit> , were in the present study differentially regulated with ageing in human skeletal muscle.
the old muscle thus exhibited higher levels of the ubiquitin-like modifier activating enzyme  <dig> , and transitional endoplasmic reticulum atpase , while the proteasome subunit beta type- <dig> , uv excision repair protein rad <dig> homolog a  and mitochondrial elongation factor tu  were down-regulated during ageing.
the vcp atpase binds multiple ubiquitin ligases and ubiquitinated proteins and triggers extraction of client proteins from complexes or cellular surfaces, often to facilitate degradation by the proteasome  <cit> .
vcp is thus central for the extraction and degradation of misfolded endoplasmic reticulum-associated   <cit>  and mitochondrial membrane proteins  <cit> .
vcp is also involved in a wide variety of cellular processes, such as dna repair  <cit> , myofibril biogenesis  <cit> , membrane fusion  <cit> , autophagosome maturation  <cit>  and mitophagy  <cit> .
the up-regulation of vcp in skeletal muscle of old women was confirmed by western-blot analysis , and is in agreement with previous observations in rat muscle  <cit> .
the scaffold protein rad23a, which was originally identified as an important factor involved in the recognition of dna lesions  <cit> , also serves as an ubiquitin receptor and plays a central role in targeting polyubiquitynated proteins, including erad substrates, for proteasomal degradation  <cit> .
tufm is important to deliver aminoacyl-trna to mitochondrial ribosome, and also promotes autophagy by interacting with autophagy-related proteins atg12–atg <dig> and atg16l <dig>  <cit> .
in addition to intracellular proteolysis, proteomics analysis of muscle ageing identified an up-regulation of the xaa-pro dipeptidase , an extracellular proteinase which specifically splits iminodipeptides with c-terminal proline or hydroxyproline.
because of the high level of iminoacids in collagen, pepd seems to be important for extracellular matrix remodeling  <cit> .
at the extracellular level, we also found that muscle ageing was associated with lower level of anti-thrombin iii , which belongs to the family of serine protease inhibitors.
with the exception of vcp  <cit> , none of these potential proteolysis biomarkers has previously been mentioned in any proteomic study on muscle ageing.
serum and transport proteins
several spots were identified as differentially expressed plasma transport proteins.
a number of these proteins were found decreased in aged muscle, including vitamin d binding protein , transthyretin  and apolipoprotein a-i .
vitamin d binding protein is a major plasma transport protein for vitamin d, while ttr transports thyroxine  and retinol  through the association with retinol-binding protein.
as a major component of the high density lipoprotein complex, apoa <dig> helps to clear fats, including cholesterol, from peripheral tissues.
apoa <dig> is also partly localized on skeletal muscle lipid droplets  <cit> .
in contrast, serotransferrin , which controls level of iron and oxidative stress by increasing iron uptake, was up-regulated in aged muscle.
another serum protein, fibrinogen gamma chain , was decreased during ageing.
altered levels of serum proteins may be related to impaired blood-flow distribution that has been reported in aged muscle fibers  <cit> .
miscellaneous
two isoforms of the es <dig> protein homolog  also decreased in aged muscle.
c21orf <dig>  is ubiquitously expressed but strongly so in heart and skeletal muscle and potential mitochondrial targeting signals were found in this protein  <cit> , but its actual physiologic function is not known.
cytosolic 5'-nucleotidase  <dig>  mainly catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates, and therefore plays an important role in both endogenous nucleoside and nucleotide pool balance  <cit> .
the age-related down-regulation of nt5c3a  that we observed in senescent women has not been previously reported in the literature.
finally, spot  <dig> decreased  <dig> -fold and is identified as apolipoprotein b mrna-editing enzyme, catalytic polypeptide-like  <dig>  that belongs to the cytidine deaminase superfamily and mediates editing of mrna.
interestingly, apobec <dig> deficiency in mice has recently been associated with a shift to a slow-fiber type in muscle, and with diminished body mass and mild myopathy  <cit> .
CONCLUSIONS
to conclude, our proteomics analyses has resulted in the identification of numerous proteins whose expression is dysregulated in old skeletal muscle.
to our knowledge, this is the most extensive proteomic study of muscle ageing in humans.
all these proteins have been classified into seven major groups, associated with myofilaments and cytoskeleton, energy metabolism, detoxification, cytoprotection, signal transduction, proteostasis and proteolysis, and figure  <dig> summarizes the functional interaction networks linking the differentially expressed proteins.
many of the candidate proteins identified in this study by differential proteomics were previously unrecognized  in previous ageing studies of skeletal muscle, in particular for proteins implicated in cytoprotection, signal transduction or proteolysis.
by comparing adult and old post-menopausal women, we have identified a group of proteins in old skeletal muscle, which indicates potential mechanisms of ageing and may lead to the development of new biomarkers of sarcopenia and novel targets for therapeutic intervention.figure  <dig>
string interaction network showing the association between differentially expressed proteins in old post-menopausal women compared to adult women.
the interaction map was generated using string  <cit>  and default settings .
the proteins names used in this network are listed in tables  <dig> and  <dig>

 ageing affects most tissues and physiologic functions, and one of the most affected organs is the skeletal muscle. the progressive decline in muscle mass and function due to ageing, which is also referred to as sarcopenia, contributes to both loss of autonomy, increased prevalence of falls, decreased resistance to metabolic aggression that increases morbidity and mortality. numerous theories have been proposed to explain muscle ageing. obviously, this is a multifactorial phenomenon which implicates intrinsic factors such as perturbations in the endocrine system, neuronal remodelling, oxidative stress and deficiencies in muscle regeneration, extrinsic factors such as diet and exercise, and also probably other unknown mechanisms. age-related degenerative changes are reflected in alterations in muscle morphology, function, and biochemical properties. muscle ageing is thus associated with muscle fiber atrophy, reduced muscle regenerative capacity, and neuropathic processes leading to motor unit denervation. mitochondrial dysfunctions with decreased capacity of oxidative enzymes and a decline of mitochondrial atp production may also be observed with ageing in skeletal muscles. the overall functional, structural, and biochemical alterations in ageing muscle have been extensively studied, but the molecular mechanisms implicated remain to be specified. the differential expression profiles of mrnas constitute a first essential level of information, but analyses of the expression profile of proteins in ageing are also required to understand the molecular mechanisms important for the muscle ageing process. in fact, unlike the genome, the proteome varies in response to many physiological or pathological factors. in addition, the proteome is orders of magnitude more complex than the transcriptome due to post-translational modifications, protein oxidation or limited protein degradation. several studies have been conducted in rat muscle and proteomic profiling has demonstrated substantial alterations in muscle proteins involved in key metabolic pathways, myofibrillar remodelling, cytoskeleton organisation and mechanisms of cytoprotection and cytodetoxification. however, few studies have been conducted with human muscle and results are contradictory. gelfi et al. have shown that several enzymes involved in oxidative metabolism, including ubiquinol-cytochrome c reductase or aspartate aminotransferase, were more abundant in elderly than in young people, while short et al. have demonstrated a decrease in these enzymes with ageing. in contrast, some results observed in rat muscle proteomic analyses were confirmed in human muscle, such as a decrease in enzymes involved in glycolytic metabolism and an increase in proteins involved in cytoprotection and cytodetoxification. because epidemiological studies have indicated accelerated muscle wasting after the fifth decade with an approximately 2% reduction in muscle mass per year, we have undertaken a top-down differential proteomic approach to determine potential changes after the fifth decade of life and to identify novel biomarkers of muscle ageing. in a previous study, we used a shot-gun proteomics approach to identify potential biomarkers. herein, we performed two dimensional gel electrophoreses using biopsies of vastus lateralis from mature adult vs. old women, and for better separation of proteins, two different strategies were conducted. firstly, overlapping immobilized ph gradient with three different ph ranges were used to improve muscle proteins separation, and secondly, we assessed low salt extracts to remove the high abundance myofibrillar proteins and improve the detection of low abundance proteins. the proteomic profiling of aged skeletal muscle fibers revealed a differential expression pattern of potential biomarkers important for energy metabolism, contractile properties, calcium signaling, cytoprotection, regulation of protein misfolding, and proteolysis. the present study demonstrates that alterations of muscle function in elderly women are associated with severe perturbed protein expression patterns and identified new potential biomarkers of sarcopenia that had not previously been described.  differentially expressed proteins during ageing in order to evaluate age-dependent alterations in the skeletal muscle proteome after the fifth decade of life, total muscle extracts from biopsies of mature adult versus old post-menopausal women were resolved by 2dge. gels with overlapping range of ipgs were used to improve the separation of total muscle extracts. 2dge with medium range ipgs revealed protein spots that were matched between all individuals. among these protein spots, were found to be differentially expressed between adult and old women, and were identified by liquid chromatography coupled to tandem mass spectrometry, corresponding to different proteins. 2dge with acidic ipgs distinguished matched protein spots, and among them were differentially expressed and identified as different proteins. narrow ipgs were also used to achieve optimal resolution in the ph - range, and this revealed a further matched spots. among these spots, statistical analysis revealed differentially expressed spots which were identified by lc-ms/ms as different proteins.figure representative 2dge image obtained from total protein extracts of human vastus lateralis skeletal muscle. 2dge was performed using a ph range of 5–, 3– or - in first dimension and sds-page in the second. protein loading was μg, and the gels were stained using colloidal coomassie blue g- differentially expressed and identified proteins are marked and spot numbers refer to table because myofibrillar proteins may hamper the detection of low abundance proteins, we also precipitated myofibrils at low ionic strength, and focused on the soluble low ionic strength extract. thirty-six gels with medium range ipgs were used to analyze lis extracts and protein spots were matched between all individuals. statistical analysis revealed that spots were differentially expressed between adult and old women. among them, were identified by lc-ms/ms, and they corresponded to different proteins. targeting the lis sub-proteome improved the 2dge analysis of the muscle proteome, as most differentially expressed lis proteins were not found in total muscle extracts.figure representative 2dge image obtained from low ionic strength extracts of human vastus lateralis skeletal muscle. 2dge was performed using a ph range of 5– in the first dimension and sds-page in the second. protein loading was μg, and the gels were stained using colloidal coomassie blue g- differentially expressed and identified proteins are marked and spot numbers refer to table interestingly, shot-gun proteomics previously identified potential biomarkers in the lis sub-proteome. it should be noted that, although shot-gun and 2dge proteomics are based on different strategies, the two techniques are complementary, as only common proteins were found in both investigations: titin, ankyrin repeat domain-containing protein, l-lactate dehydrogenase β, furamate hydratase, fatty acid binding protein and carbonic anhydrase. overall in the present 2dge study, protein spots were matched between all individuals, were differentially expressed and identified, and they corresponded to different proteins. table and table summarize the main properties of the proteins differentially regulated in skeletal muscle between adult and old post-menopausal women. absolute fold changes varied from to. even though some fold-changes appeared relatively small, there were parallel changes of several components in each pathway or biological process, suggesting some important coordinated and specific regulations.table differentially expressed muscle proteins in total extract between adult and old post-menauposal women perturbations of the myofilament network and cytoskeleton with ageing sarcomeric proteins muscle contraction is generated by an interaction between the molecular motor myosin and filamentous actin. myosin is a hexameric protein that consists of two heavy chain subunits, two alkali light chain subunits and two regulatory light chain subunits. myosin light chains typically exhibit various isoforms, and in our study, muscle ageing was associated with higher level of one myosin light chain 1/ skeletal muscle isoform and lower level of one myosin regulatory light chain ventricular/cardiac muscle isoform. perturbations in myofibrillar contractile proteins were confirmed by the up-regulation of one isoform of fast troponin t which is a major regulator of the thin filament. troponin t directly interacts with key components in the thin filament regulatory system to mediate the activation and force development of actomyosin contractile units. age-related changes in fibers expressing various myosin light chain isoforms were previously described for myl myl and tnnt; however, these changes were manifested differentially in distinct muscles. interestingly, the old skeletal muscle also exhibited altered levels of several fragments of sarcomeric proteins. thus, there were higher levels of n-terminal and c-terminal fragments of skeletal α-actin, and a higher level of a c-terminal fragment of myosin-, the type iix adult fast myosin heavy chain. the old muscle also exhibited lower level of a c-terminal fragment of ttn, when compared to adult muscle. such fragments were not observed for non-sarcomeric proteins, and may indicate perturbations in sarcomeric proteolytic pathways. a feature of human muscle ageing was also the increased expression of ankyrin repeat domain-containing protein. ankrd is a member of the mechano-sensing proteins that link myofibrillar stress response to muscle gene expression. ankrd interacts both with i-band sarcomeric proteins and with nuclear transcription factors. this stretch-response protein is preferentially expressed in slow type-i fibers, and is induced by denervation, which is consistent with neuronal remodeling in ageing muscle. aging of skeletal muscle involves a decrease in both total number and size of muscle fibers. no consensus has been reached in the literature on whether hypoplasia is associated with a shift in fiber type distribution towards higher, lower or unaltered percentages of type-i versus type-ii fibers. however, previous studies based on myosin heavy chain histochemistry agreed that atrophy mostly affects type-ii fibers. in our 2dge analyses, there was no clear evidence for a fast-to-slow transition on the unique basis of the differential expression of myosin light chain and troponin isoforms. cytoskeletal proteins the integrity of muscle fibers depends on cytoskeletal components, which align sarcomeres and anchor them across the sarcolemma to the basement membrane. two isoforms of vinculin were enhanced during ageing. vcl localizes to adhesion junctions and is a central component of muscle costameres. vcl is placed between the integrin-talin complex and the actin cytoskeleton and is a major candidate for transduction of force during the contractile cycle. besides binding to actin, vcl interacts with signaling networks and is important for signal transduction between the extracellular matrix and the cytoskeleton. finally, the four and a half lim domain protein, which is down-regulated in our analysis, is an adaptor protein with numerous interaction partners. in adult fibers, fhl interacts with cytoskeletal actin and co-localizes with integrin receptors at the periphery of z-discs. by binding to integrin and actin, fhl might then directly link the cytoskeleton to the extracellular matrix. fhl also plays a role in myogenic progenitor cells, where it localizes to the nucleus and has been implicated in the regulation of proliferation and differentiation. vcl and fhl have not been previously identified by proteomic surveys of human skeletal muscle ageing. shot-gun proteomics confirmed the up-regulation of ankrd, and western blotting experiments performed with total muscle extracts confirmed the differential expressions that we observed for vcl and fhl in lis extracts. in all, the modifications that we report for sarcomeric actomyosin and cytoskeletal proteins are most likely related to disorganization of myofibers in old muscles. moreover, these changes suggest an influence of the ageing process on the maintenance of the proper organization of sarcolemma in regular structures that are closely linked to costameres, and on the response to extracellular signals. thus, the age-related changes in sarcomeric and cytoskeletal proteins may result in alterations in contractile properties and contribute to the development of sarcopenia.figure examples of differential expression of cytoskeletal proteins. representative sections of 2dge images and representative western blots for vinculin and four and a half lim domains. in each panel, histograms represent normalized volume of protein spot, and western blot quantification for adult and old post-menopausal women. results are indicated as means ± se. *: p < indicates significant difference between adult and old women. age-related changes in skeletal muscle affect signal transduction in skeletal muscle, intracellular calcium is an important secondary messenger for signal transduction and is essential for cellular processes such as excitation-contraction coupling. action potentials elicit contractions by releasing ca2+ from the sarcoplasmic reticulum via the ryanodine receptors. ryrs are modulated directly or indirectly by various ions, small molecules and proteins, including calsequestrin. in this study, proteomics analysis of muscle ageing identified an up-regulation of calsequestrin-, which is a major intra-sr ca2+ buffer that regulates the activity of ryrs. ryr receptors have several potential phosphorylation sites in their cytoplasmic domains and protein kinase a has been shown to phosphorylate ryrs. our analysis shows an increased level of the pka type iα regulatory subunit during ageing. among other substrates pka can phosphorylate ryr, and pka-mediated phosphorylation of ryr may result in leaky ryr channels and impaired ca2+ homeostasis. after initiation of muscle contraction by increasing cytoplasmic ca2+, ca2+ is pumped back to the sr by sarcoplasmic reticulum ca2+ atpase leading to relaxation. sarcalumenins are major luminal glycoproteins that codistribute with serca and play a role in ca2+ transport and sequestration. there are two srl isoforms that are generated by alternative splicing. in the present study, the 53-kda isoform was found to be reduced in aged human muscle, and this reduction was confirmed by western-blotting. the age-related decrease of srl is in agreement with previous studies in rat muscle indicating a shorter half-life of the 53-kda isoform and a lower level of the 160-kda isoform.figure examples of differential expression of proteins implicated in signal transduction. representative sections of 2dge images and representative western blots for sarcalumenin, myozenin-, annexin a, annexin a and glutathione s-transferase omega-. in each panel, histograms represent normalized volume of protein spot, and western blot quantification for adult and old post-menopausal women. results are indicated as means ± se. *: p < indicates significant difference between adult and old women. we have also identified an up-regulation of myozenin- which is expressed in fast-twitch fibers of skeletal muscle and modulates the function and substrate specificity of calcineurin, a ca2+/calmodulin-dependent serine-threonine phosphatase that plays an important role in transducing calcium-dependent signals. this increase in myoz was also confirmed by western-blotting. in skeletal muscle, numerous proteins can bind ca2+, and muscle ageing was further associated with higher level of three members of the annexin family which undergo ca2+-dependent binding to the cellular membranes. we identified annexin a, a and a. annexins have been involved in a broad range of molecular and cellular processes. noteworthy anxa may contribute to the regeneration of skeletal muscle tissue by modulating migration and fusion of satellite cells. anxa is overexpressed in different muscular dystrophies and can further participate in sarcolemmal and t-tubular repair processes. anxa also promotes membrane repair by self-assembling into two-dimensional arrays on membranes. anxa was originally described as a protein that provokes fusion of lipid vesicules. more recently, anxa was identified as an essential protein for autophagy induction by modulating the intracellular ca2+ concentration. western-blotting experiments confirmed the overexpression of anxa and anxa in total extracts of old muscle, compared to adult muscle. glutathione s-transferase omega- is distinguished from the other glutathione s-transferase family members by a different active center amino acid residue, which results in loss of prototypical glutathione conjugating activity. instead, human gsto is reported to potentiate skeletal muscle ryanodine receptor. the age-related down-regulation of gsto was confirmed by western-blot, and may thereby be involved in the impairment of ca2+ homeostasis. most of these signal transduction proteins have never been reported in previous proteomic studies of muscle ageing. overall, our findings suggest significant alterations in ca2+ signaling which may be important for the age-related modifications in muscle contractile properties and may contribute to muscle weakness. perturbations in the energy metabolism of old muscle disturbance in energy metabolism is another characteristic feature of old muscles. decreased activities of glycolytic enzymes were previously reported in rat and in human skeletal muscles, whereas expression of mitochondrial enzymes was more controversial in the literature. our study points to an age-associated decline in key enzymes of the glycolytic, krebs cycle and oxidative phosphorylation pathways. cytoplasmic energy metabolism in the current study, perturbations in the energy metabolism of old muscle were indicated by the down-regulation of five isoforms of monomeric creatine kinase. sds-stable dimeric and trimeric forms of ckm were previously described in mice muscle, and we also observed age-related decreases in dimeric and trimeric forms of ckm. creatine/phosphocreatine is central to maintain energetic homeostasis as it connects intracellular sites of energy demand with sites of atp production. at these sites, ckm catalyses the transphosphorylation between phosphocreatine and adp. in sarcomeric m-line, ckm interacts with myomesin and supplies atp for the actomyosin contractile unit. the first step of glycogenolysis pathway is the production of glucose-6-phosphate by glycogen phosphorylase, which catalyzes the phosphorolytic cleavage of a glucosyl residue from the glycogen polymer. four isoforms of pygm increased, and one decreased with ageing. the resulting glucose 1-phosphate molecule is converted by phosphoglucomutase to glucose 6-phosphate. proteomic analysis revealed a decreased expression of phosphoglucomutase- in elderly women. several glycolytic enzymes were also down-regulated with ageing. enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate. in the muscle of post-menopausal women, four isoforms of the muscle specific β-enolase were selectively decreased during ageing. we also detected lower levels of ldhb which catalyzes the inter-conversion of pyruvate and lactate with concomitant inter-conversion of nadh and nad+. finally, down-regulations of glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase 1-like protein were observed. cytosolic gpd together with its mitochondrial isoform, constitute the gpd shuttle, which is essential for mitochondrial oxidation of glycolytic nadh. although less active than its gpd counterpart, gpd1l exhibits dehydrogenase activity, and is also implicated in the regulation of hypoxia. age-related decline in cytoplasmic glycerol-3-phosphate dehydrogenase may indicate reduced mitochondrial oxidation of cytosolic nadh in old muscle. triosephosphate isomerase, that catalyzes the isomerization of the dihydroxyacetone phosphate and d-glyceraldehyde 3-phosphate, was the only glycolytic enzyme more abundant in old muscle. tpi ensures that dhap produced by aldolase is further metabolized by the glycolytic enzymes. impairment of tpi can result in chemical conversion of dhap into toxic methylglyoxal promoting the formation of advanced glycation end-products. in the old muscle, an increased level of tpi may then represent a compensatory adaptation to avoid excessive formation of toxic products. mitochondrial energy metabolism alterations in the mitochondrial krebs cycle were revealed by the age-dependent reduction in two subunits of the pyruvate dehydrogenase, pyruvate dehydrogenase e component subunit β and dihydrolipoyl dehydrogenase, and by the reduction in aconitate hydratase and fh, two enzymes that catalyze the isomerization of citrate to isocitrate and the hydration of furamate to malate, respectively. moreover, alterations in oxidative phosphorylation were also revealed by lower levels of three components of the respiratory chain, nadh dehydrogenase iron-sulfur protein of complex i, cytochrome b-c complex subunit rieske of complex iii and subunit 5a of cytochrome c oxidase of complex iv. ageing was further associated with lower level of atp synthase subunit β. among these proteins implicated in energy metabolism, two have never been reported in previous muscle ageing studies, and fh and atp5b were similarly found in our shot-gun experiment. western blotting experiments confirmed alterations in glycolytic and oxidative metabolism, as we observed decreased expression of eno gpd ndufs and uqcrfs in total extracts of old muscle compared to adult muscle. concomitant decreases of key enzymes of krebs cycle and major complexes of oxidative phosphorylation provided evidences for alteration of mitochondrial metabolism in the old skeletal muscle.figure examples of differential expression of proteins implicated in energy metabolism. representative sections of 2dge images and representative western blots for β-enolase, glycerol-3-phosphate dehydrogenase, nadh dehydrogenase fe-s protein and cytochrome b-c complex subunit rieske. in each panel, histograms represent normalized volume of protein spot, and western blot quantification for adult and old post-menopausal women. results are indicated as means ± se. *: p < indicates significant difference between adult and old women. lipid metabolism in addition to glucose, lipids are another source of energy in skeletal muscle. several intracellular fatty acid-binding proteins have been identified. they have important functions in the transport of intracellular fatty acids by increasing their solubility and have been shown to enhance the transport of fatty acids from the cell membrane to the site of oxidation, i.e. the mitochondria, and to the site of esterification into intramyocellular triglycerol. muscle ageing was associated with the down-regulation of heart fabp, the major fabp in skeletal muscle, and the up-regulation of adipocyte fabp. fabp plays an important, but merely permissive role in fatty acid uptake by skeletal muscles. fabp is an adipocyte marker, but is also expressed in muscle fibers. therefore, the age-related up-regulation of fabp may relate to increased number of adipocytes, and/or to an increased expression in muscle fibers. except in our shot-gun study, fabp was not noticed in previous reports on sarcopenia. of note, the lipid chaperone fabp was also identified as a predominant positive regulator of toxic lipid-induced endoplasmic reticulum stress, and fabp up-regulation could suggest increased er stress in old muscle. overall, our proteomic analyses therefore provided strong evidences for a decline in both glycolytic and mitochondrial energy metabolism in the old skeletal muscle. declines in mitochondrial oxidative capacity were previously reported with advancing age, although physical activity, rather than chronological age, was also reported to be the primary determinant. because the activity score was significantly higher for the adult than for the old women group, future studies will be required to specify whether mitochondrial oxidative capacity still decreases in old subjects maintaining physical activity scores. detoxification of cytotoxic products and cytoprotection in the old muscle protection against mitochondrial oxidative stress mitochondrial dysfunctions may lead to an excessive production of reactive oxygen species, and accumulating evidences suggest that oxidative stress underlies the ageing process in skeletal muscle. the removal of h2o in cells is mediated by catalase, glutathione peroxidase and peroxiredoxin. in the present study, ageing of human skeletal muscle was associated with lower levels of cat and higher levels of prdx. because cat is localized preferentially in peroxisomes, its function is limited to the inactivation of h2o diffusing into these organelles. in contrast, prdx is the only peroxiredoxin restricted to mitochondria, and high levels of prdx may provide a primary line of defence against h2o over-produced by the respiratory chain in old muscle mitochondria. neither cat nor prdx have previously been reported in proteomic analyses of muscle ageing. a wide range of activities have been reported for protein dj-, however, there is consensus that park is responsive and protective against mitochondrial oxidative stress. park is an atypical peroxiredoxin-like peroxidase. during oxidative attack, park is relocalized to mitochondria, has a functional role in scavenging mitochondrial h2o and decreases mitochondrial fragmentation. therefore elevated levels of park in the old muscle may be important in regulating cellular antioxidant capacity. detoxification of cytotoxic products oxidative stress increases the production of cytotoxic aldehydes, which can react with cellular proteins, nucleic acids and cell membranes. protection against reactive aldehydes is provided by several families of detoxification enzymes, including aldo-keto reductase and aldehyde dehydrogenase. our proteomic analysis provided evidences for perturbed scavenging of reactive aldehyde products in the old muscle, as aldehyde dehydrogenase and alcohol dehydrogenase, were up-regulated, while aldose reductase and delta-1-pyrroline-5-carboxylate dehydrogenase decreased with ageing. similar regulations were previously reported during ageing in rat skeletal muscle for aldh and akr1b. however, no previous study of muscle ageing has identified the differential expression of akr1a and aldh4a the mitochondrial enzyme aldh detoxifies aromatic and aliphatic aldehydes, which are produced during oxidative stress as a result of lipid peroxidation. the cytosolic oxidoreductase akr1a has broad substrate specificity, and similarly catalyzes the reduction of aliphatic and aromatic aldehydes, ketones, and xenobiotics. the up-regulations of aldh and akr1a thus suggest enhanced scavenging of reactive aldehyde products in the old skeletal muscle. our western blot analyses confirmed that muscle ageing is associated with higher levels of aldh.figure examples of differential expression of proteins implicated in cytoprotection, cytodetoxification or proteolysis. representative sections of 2dge images and representative western blots for aldehyde dehydrogenase, α-crystallin b chain, heat shock kda protein 1a/1b, hsp 90-beta, and transitional endoplasmic reticulum atpase. in each panel, histograms represent normalized volume of protein spot, and western blot quantification for adult and old post-menopausal women. results are indicated as means ± se. *: p < indicates significant difference between adult and old women. while aldh and akr1a act principally as detoxification enzymes, akr1b and aldh4a have additional roles besides that of detoxification. accumulating evidence attributes a significant role to akr1b in transducing cytotoxic signals initiated by inflammatory cytokines. akr1b inhibitors have been reported to disrupt signalling cascades leading to nfkb activation, and reduced levels of akr1b may therefore be important to limit activation of the nfkb pathway in the old muscle. aldh4a amino acid sequence diverges from the other aldh and this enzyme is mostly involved in metabolic regulation. aldh4a is metabolically important, since its substrate appears as a common intermediate in the degradative and biosynthetic pathways of the amino acids arginine, citrulline, ornithine and proline to and from glutamic acid. the down regulation of aldh4a may thus indicate reduced intermediary metabolism in the old skeletal muscle. quality control of cellular proteins to detect, refold, and eventually eliminate abnormal proteins, cells use quality control mechanisms that buffer protein homeostasis against cellular stress. the proteomic analysis described here demonstrated the differential regulation of spots identified as heat shock proteins. all were increased with ageing and they encompassed hsp groups in human: hspa, hspb and hspc. the small heat shock proteins are atp-independent chaperones. hspbs prevent the aggregation of improperly folded or partially denatured proteins, and are involved in their transfer to the atp-dependent chaperones or to the protein degradation processes such as proteasomes or autophagosomes. three hspbs displayed higher levels in old women compared to adult women: heat shock protein beta-, alpha-crystallin b chain and heat shock protein beta-. similar up-regulation of hspb hspb and hspb have been previously reported for rat muscle ageing. our western blot experiments performed with total muscle extracts confirmed that the old human muscle exhibited higher levels of hspb than adult muscle. in addition to chaperone activities, hspb and hspb have the ability to control the redox status, protect the actin cytoskeleton and inhibit apoptotic cell death. the atp-dependent chaperones of the hspa group are essential for proteostasis as they contribute to the folding and assembly of nascent polypeptides, the transport of proteins across membranes, and the selection of misfolded proteins for degradation. muscle ageing was associated with the up-regulation of two hspa proteins: the heat shock kda protein 1a/1b, and the mitochondrial stress- protein. none of these hspa have been previously reported in proteomic analyses of muscle ageing, and our western blot experiments confirmed the age-related up-regulation of hspa1a. hspa1a is the most abundant inducible cytoprotective hsp chaperone. notably in rodents, overexpression of hspa1a has been repeatedly reported to attenuate muscle atrophy induced by immobilization, lengthening contractions or cryo-lesions. it is therefore likely that the old human muscle may increase hspa1a levels as a protection against muscle atrophy. hspa is a central component of the mitochondrial protein import motor and it plays a key role in the folding of matrix-localized mitochondrial proteins. hspa is the only known mitochondrial hsp chaperone, and thus serves as a unique scavenger of toxic protein aggregates in human mitochondria. one hspc was further found to be up-regulated in these ageing muscles: the heat shock protein hsp. hsp which has not been identified in previous muscle ageing studies, is another important chaperone that functions downstream of hspa in the atp-dependent folding and conformational regulation of many client proteins, including protein kinases, steroid receptors, endothelial no synthase and transcription factors. most of the clients, with which hsp interacts, are thus involved in signal transduction, making hsp a critical factor in cell signalling. western blot experiments confirmed the age-related up-regulation of hsp 90-beta. finally and in addition to the hsp proteins, the chaperone operating in the endoplasmic reticulum, protein disulfide-isomerase a, was also found to increase with human muscle ageing, and this observation is in agreement with previous studies in rat. pdia is a multifunctional protein with thiol-protein disulphide oxidoreductase activity, which ensures proper folding of glycoproteins and assembly of major histocompatibility complex class i complex. outside the er, pdia also functions as a plasma membrane receptor for 1α,25-dihydroxy vitamin d, which might be important with regard to the implication of vitamin d in ageing. cytoprotection in the old skeletal muscle in agreement with a previous study in rat, higher levels of two isoforms of carbonic anhydrase were identified in senescent human muscle. no consensus has been reached in the literature about ca as both increased and decreased levels were observed in old muscles. our analyses of muscle ageing in women indicated an overexpression of isoforms of ca and this was confirmed by our shot-gun study. the various isoforms of cas play a crucial role in co2-removal and co2-provision for metabolic processes, and are central for the acid–base balance and the regulatory processes of ion homeostasis. numerous proteins are involved in ion homeostasis, including the selenium-binding protein of which two isoforms were increased and one isoform was decreased in ageing. proteolytic systems muscle proteins are continuously turning over, and cells contain multiple proteolytic systems to carry out the degradation process. several components of the major proteolytic systems, i.e. ubiquitin-proteasome and lysosomal autophagy, were in the present study differentially regulated with ageing in human skeletal muscle. the old muscle thus exhibited higher levels of the ubiquitin-like modifier activating enzyme, and transitional endoplasmic reticulum atpase, while the proteasome subunit beta type-, uv excision repair protein rad homolog a and mitochondrial elongation factor tu were down-regulated during ageing. the vcp atpase binds multiple ubiquitin ligases and ubiquitinated proteins and triggers extraction of client proteins from complexes or cellular surfaces, often to facilitate degradation by the proteasome. vcp is thus central for the extraction and degradation of misfolded endoplasmic reticulum-associated and mitochondrial membrane proteins. vcp is also involved in a wide variety of cellular processes, such as dna repair, myofibril biogenesis, membrane fusion, autophagosome maturation and mitophagy. the up-regulation of vcp in skeletal muscle of old women was confirmed by western-blot analysis, and is in agreement with previous observations in rat muscle. the scaffold protein rad23a, which was originally identified as an important factor involved in the recognition of dna lesions, also serves as an ubiquitin receptor and plays a central role in targeting polyubiquitynated proteins, including erad substrates, for proteasomal degradation. tufm is important to deliver aminoacyl-trna to mitochondrial ribosome, and also promotes autophagy by interacting with autophagy-related proteins atg12–atg and atg16l. in addition to intracellular proteolysis, proteomics analysis of muscle ageing identified an up-regulation of the xaa-pro dipeptidase, an extracellular proteinase which specifically splits iminodipeptides with c-terminal proline or hydroxyproline. because of the high level of iminoacids in collagen, pepd seems to be important for extracellular matrix remodeling. at the extracellular level, we also found that muscle ageing was associated with lower level of anti-thrombin iii, which belongs to the family of serine protease inhibitors. with the exception of vcp, none of these potential proteolysis biomarkers has previously been mentioned in any proteomic study on muscle ageing. serum and transport proteins several spots were identified as differentially expressed plasma transport proteins. a number of these proteins were found decreased in aged muscle, including vitamin d binding protein, transthyretin and apolipoprotein a-i. vitamin d binding protein is a major plasma transport protein for vitamin d, while ttr transports thyroxine and retinol through the association with retinol-binding protein. as a major component of the high density lipoprotein complex, apoa helps to clear fats, including cholesterol, from peripheral tissues. apoa is also partly localized on skeletal muscle lipid droplets. in contrast, serotransferrin, which controls level of iron and oxidative stress by increasing iron uptake, was up-regulated in aged muscle. another serum protein, fibrinogen gamma chain, was decreased during ageing. altered levels of serum proteins may be related to impaired blood-flow distribution that has been reported in aged muscle fibers. miscellaneous two isoforms of the es protein homolog also decreased in aged muscle. c21orf is ubiquitously expressed but strongly so in heart and skeletal muscle and potential mitochondrial targeting signals were found in this protein, but its actual physiologic function is not known. cytosolic 5'-nucleotidase mainly catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates, and therefore plays an important role in both endogenous nucleoside and nucleotide pool balance. the age-related down-regulation of nt5c3a that we observed in senescent women has not been previously reported in the literature. finally, spot decreased -fold and is identified as apolipoprotein b mrna-editing enzyme, catalytic polypeptide-like that belongs to the cytidine deaminase superfamily and mediates editing of mrna. interestingly, apobec deficiency in mice has recently been associated with a shift to a slow-fiber type in muscle, and with diminished body mass and mild myopathy.  to conclude, our proteomics analyses has resulted in the identification of numerous proteins whose expression is dysregulated in old skeletal muscle. to our knowledge, this is the most extensive proteomic study of muscle ageing in humans. all these proteins have been classified into seven major groups, associated with myofilaments and cytoskeleton, energy metabolism, detoxification, cytoprotection, signal transduction, proteostasis and proteolysis, and figure  summarizes the functional interaction networks linking the differentially expressed proteins. many of the candidate proteins identified in this study by differential proteomics were previously unrecognized in previous ageing studies of skeletal muscle, in particular for proteins implicated in cytoprotection, signal transduction or proteolysis. by comparing adult and old post-menopausal women, we have identified a group of proteins in old skeletal muscle, which indicates potential mechanisms of ageing and may lead to the development of new biomarkers of sarcopenia and novel targets for therapeutic intervention.figure string interaction network showing the association between differentially expressed proteins in old post-menopausal women compared to adult women. the interaction map was generated using string and default settings. the proteins names used in this network are listed in tables and 
this study describes the most extensive proteomic analysis of muscle ageing in humans, and identified new potential biomarkers. in addition to total muscle extracts, low-ionic strength extracts were investigated to remove high abundance myofibrillar proteins and improve the detection of low abundance proteins. in order to better understand the molecular mechanisms underlying muscle chronological ageing in human, we have undertaken a top-down differential proteomic approach to identify novel biomarkers after the fifth decade of age. notably, many of the differentially expressed proteins were central for proteostasis, including heat shock proteins and proteins involved in proteolysis. two-dimensional gel electrophoreses with overlapping ipgs were used to improve the separation of muscle proteins. our results suggested important modifications in cytosolic, mitochondrial and lipid energy metabolism, which may relate to dysfunctions in old muscle force generation. a fraction of the differentially expressed proteins were linked to the sarcomere and cytoskeleton, which may account for alterations in contractile properties. none of them were previously recognized as differentially expressed in old muscles, and each may represent a novel starting point to elucidate the mechanisms of muscle chronological ageing in humans. overall, protein spots were matched between all individuals, were differentially expressed and identified by mass spectrometry, and they corresponded to different proteins. in line with muscle contraction, we also identified proteins related to calcium signal transduction. muscle ageing was further characterized by the differential regulation of several proteins implicated in cytoprotection, ion homeostasis and detoxification.
 muscle ageing contributes to both loss of functional autonomy and increased morbidity. muscle atrophy accelerates after years of age, but the mechanisms involved are complex and likely result from the alteration of a variety of interrelated functions. in order to better understand the molecular mechanisms underlying muscle chronological ageing in human, we have undertaken a top-down differential proteomic approach to identify novel biomarkers after the fifth decade of age.  muscle samples were compared between adult and old post-menopausal women. in addition to total muscle extracts, low-ionic strength extracts were investigated to remove high abundance myofibrillar proteins and improve the detection of low abundance proteins. two-dimensional gel electrophoreses with overlapping ipgs were used to improve the separation of muscle proteins. overall, protein spots were matched between all individuals, were differentially expressed and identified by mass spectrometry, and they corresponded to different proteins. our results suggested important modifications in cytosolic, mitochondrial and lipid energy metabolism, which may relate to dysfunctions in old muscle force generation. a fraction of the differentially expressed proteins were linked to the sarcomere and cytoskeleton, which may account for alterations in contractile properties. in line with muscle contraction, we also identified proteins related to calcium signal transduction. muscle ageing was further characterized by the differential regulation of several proteins implicated in cytoprotection, ion homeostasis and detoxification. notably, many of the differentially expressed proteins were central for proteostasis, including heat shock proteins and proteins involved in proteolysis.  this study describes the most extensive proteomic analysis of muscle ageing in humans, and identified new potential biomarkers. none of them were previously recognized as differentially expressed in old muscles, and each may represent a novel starting point to elucidate the mechanisms of muscle chronological ageing in humans. keywords humanskeletal muscleageingsarcopeniaproteomicsbiomarkersissue-copyright-statement© the author 2014 