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For citation purposes: Ohlendieck K. Proteomics of exercise-induced skeletal muscle adaptations. OA Sports Medicine 2013 Mar 01;1(1):3.
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Ana
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Cell
Biol
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Proteomics of exercise-induced skeletal muscle adaptations
K Ohlendieck*
AbstractIntroductionThe systems biological analysis of dynamic protein constellations an-d the determination of proteome-wide alterations due to physiolog-ical adaptations play an increasin-g role in modern sports medicine. Several large-scale studies on the effect of physical training in hum-ans and relevant animal models have decisively improved our glo-bal understanding of the molecul-ar and cellular mechanisms invol-ved in skeletal muscle changes d-uring exercise. The aim of this cr-itical review was to discuss the proteomics of exercise-induced sk-eletal muscle adaptations. DiscussionBuilding on this extensive knowl-edge of conventional exercise bio-logy, refined protein biochemical and mass spectrometric technolo-gies can now be employed to stu-dy subtle changes in protein con-centration, isoform expression pa-tterns, protein–protein interactions and/or post-translational modifica-tions following physical activity. Besides being a key method for t-he elucidation of fibre plasticity and muscle transformation, the s-ystematic application of mass spe-ctrometry-based proteomics prom-ises to play a prevalent role in t-he establishment and evaluation of preventative exercise regimes to counteract skeletal muscle was-ting and metabolic disturbances in common
disorders with muscular involve-ment such as diabetes, obesity, car-diovascular disease, cancer cachexia or sarcopenia of old age. In this criti-cal review, the impact of recent pro-teomic profiling studies of physical exercise is examined and its implica-tions for our molecular understand-ing of skeletal muscle adaptations are discussed.ConclusionRecent findings from mass spec-trometry-based proteomic studies of physical exercise have identified a variety of adaptive changes in mus-cle proteins involved in cellular sig-nalling, fibre contraction, metabolic pathways and the cellular stress re-sponse. The establishment of these novel biomarkers, which are charac-teristic for exercise-related muscle adaptations, will be extremely useful for the detailed biochemical evalua-tion of physical training programs.
IntroductionOver the last decade, a large num-ber of scientific breakthroughs have transformed the field of exercise bi-ology1. Our understanding of gene regulation and protein alterations in response to physical exercise has dramatically improved through the application of molecular and cel-lular analyses of skeletal muscle adaptations. This has involved the elucidation of novel structural, func-tional and metabolic aspects during force generation and physiological adaptability in response to different training regimes2. Exercise triggers diverse physiological stimuli that involve neuronal, mechanical, meta-bolic and hormonal signals that are sensed, transduced and integrated in a highly coordinated manner3.
The repeated recruitment of specific muscle groups causes lasting altera-tions in gene expression patterns and distinct changes in the concen-tration, isoform repertoire and/or post-translational modifications of skeletal muscle proteins4.
During muscle adaptations, a crucial relationship exists between contraction-induced signalling cas-cades and downstream effects in contractile fibres on the level of gene activation, mRNA processing, protein synthesis and protein assembly, as well as metabolic regulation. Novel integrative approaches attempt to study these effects of exercise-in-duced physiological disturbances on the level of the genome, tran-scriptome, proteome and metabo-lome5-7. In this article, the findings from recent proteomic studies that have focused on large-scale analyses of exercise-induced changes in the protein complement from skeletal muscle are reviewed. An attempt is made to assess how these molecular findings can now be used to rational-ize the physiological and biochemical basis of muscle adaptations and to suitably plan future global studies in exercise biology.
DiscussionThe author has referenced some of his own studies in this review. These referenced studies have been con-ducted in accordance with the Dec-laration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics com-mittees related to the institution in which they were performed. All hu-man subjects, in these referenced studies, gave informed consent to participate in these studies.
* Corresponding author Email: [email protected]
Muscle Biology Laboratory, Department of Biology, National University of Ireland, May-nooth, Co. Kildare, Ireland
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For citation purposes: Ohlendieck K. Proteomics of exercise-induced skeletal muscle adaptations. OA Sports Medicine 2013 Mar 01;1(1):3.
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cleavage of some polypeptide chains into more than one subunit, as well as an extremely large variety of post-translational modifications9.
The ultimate goal of applying a sys-tems biological approach to the field of applied myology is the establish-ment of a unifying scheme that ex-plains how the metabolic status and a plethora of physiological stimuli from extracellular and intramuscu-lar systems result in functional and structural adaptations to enhanced neuromuscular activity4. Figure 1 outlines the relationship between muscle activity-induced signalling, the integration of these physiological
dynamic nature of protein expres-sion patterns8. In contrast to the relatively stable genome, the global protein constellation of specific cell types or tissues is highly variably and constantly adapting to changed functional demands and environ-mental influences. The discrepancy between the extremely large num-ber of individual protein species in the human body and the much lower number of identified genes is due to various regulatory mechanisms and extensive protein conversions. This includes the existence of alternative promoter repertoires, the alternative splicing of mRNAs and the enzymatic
From genome to muscle proteomeFollowing the elucidation of the hu-man genome and a variety of animal model genomes of physiological or pathological relevance, a major chal-lenge in the field of biomedicine is now presented by the determina-tion of the cell-specific activation of all identified genes and the many functions of their expressed protein products. The biochemical charac-terization of the proteins encoded by the approximately 20,300 human genes is complicated by the multi-functionality of many protein mole-cules, the highly diverse interactions within protein complexes and the
Figure 1: Schematic overview of the relationship between muscle activity-induced cellular signalling, the integration of various physiological stimuli during enhanced neuromuscular activity and how these physiological alterations may influence contractile fibres on the level of the genome, transcriptome, proteome and metabolome.
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For citation purposes: Ohlendieck K. Proteomics of exercise-induced skeletal muscle adaptations. OA Sports Medicine 2013 Mar 01;1(1):3.
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involved in gel electrophoresis-based proteomic studies of muscle adapta-tions during exercise. Alternatively, proteins can be separated by liquid chromatography or a combination of chromatographical and electro-phoretic methodology. Following the preparation of total protein extracts or the separation of individual or-ganelles, components from varying proteomes can be differentially la-belled with fluorescent dyes prior to gel electrophoresis. The fluorescence difference in-gel electrophoretic
alterations in protein density, protein localization, protein isoform expres-sion, protein–protein interactions and post-translational modifications.
Proteomic profiling of muscle biopsiesIn order to study the accessible pro-teome from skeletal muscle tissues, highly efficient methods for extrac-tion, fractionation, separation and detection of proteins have to be com-bined in a rationalized workflow10. Figure 2 summarizes the main steps
stimuli and changes on the level of the genome, transcriptome, proteome and metabolome. Importantly, since contractile fibres and its associated nerves, capillaries, connective tissue layers and satellite cells represent highly complex physiological sys-tems, its functional behaviour during an altered physiological state may be useful for establishing the molecular mechanisms that underlie the plas-ticity of the physiome. A crucial part of the physiological dynamics of the neuromuscular system is based on
Figure 2: Summary of the main preparative and analytical steps involved in gel electrophoresis-based proteomic studies of muscle adaptations during physical exercise (DIGE, difference in-gel electrophoresis; IEF, isoelectric focusing; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis).
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For citation purposes: Ohlendieck K. Proteomics of exercise-induced skeletal muscle adaptations. OA Sports Medicine 2013 Mar 01;1(1):3.
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acid sequence of individual peptides compared to databanks containing sequence information of the human proteome. The peptide information resulted in a 20% sequence cover-age, which unequivocally identified the major protein species contained in the 2D spot of interest as the slow TNNT1 isoform of troponin TnT from human skeletal muscle.
Exercise proteomicsIn the field of sports medicine, the application of mass spectrometry-based proteomics attempts to iden-tify global mechanisms of protein alterations that support the estab-lishment of the endurance phenotype or power performance5–7. Exercise proteomics was used to study pro-tein alterations in humans13–18 and animal models of physical activ-ity19–28, as summarized in Table 1. This has included the analysis of human vastus lateralis muscle in re-sponse to interval training using both 2D gel electrophoresis and the quan-titative isobaric tags for relative and absolute quantitation (iTRAQ) meth-od13, the mitochondrial proteome from human vastus lateralis muscle
include elements involved in neuromuscular transmission, exci-tation–contraction coupling, ion ho-meostasis, signal transduction, fibre assembly, contraction, relaxation, fibre elasticity, cytoskeletal main-tenance, metabolic integration, me-tabolite transportation, glycolysis, fatty acid oxidation, citric acid cycle, oxidative phosphorylation, lipid me-tabolism, nucleotide metabolism, gene regulation, transcription, trans-lation, protein synthesis, protein as-sembly, protein storage, fibre repair, neogenesis, immune response, de-toxification and the cellular stress response12. Figure 4 highlights the various steps in the proteomic iden-tification of a key protein of the con-tractile apparatus, the slow isoform of troponin TnT. Shown is the unique position of this troponin subunit in a two-dimensional gel of human vastus lateralis muscle, based on its molecular mass of approximately 30 kDa and its isoelectric point of pI 6.4. Following excision of the protein spot and its controlled proteolytic digestion by trypsination, the gener-ated peptide population is analysed by mass spectrometry and the amino
(DIGE) method can visualize sev-eral thousand muscle proteins in a single analytical experiment, mak-ing it an extremely valuable tool for comparative proteomics11. Fluores-cently tagged proteins are routinely separated by high-resolution two-dimensional gel electrophoresis us-ing isoelectric focusing in the first dimension and sodium dodecyl sul-phate polyacrylamide slab gel elec-trophoresis in the second dimension. Following the densitometric analy-sis of spot patterns, proteins of in-terest are excised and digested for mass spectrometric peptide analysis. Peptide sequences are compared to international databanks for the un-equivocal identification of individual protein species.
The differential expression of identified protein candidates is then usually verified by immunoblotting surveys. The biochemical, cell bio-logical and physiological characteri-zation of novel proteins is routinely carried out by enzyme assays, bind-ing tests, confocal microscopy and functional analyses10. As summarized in Figure 3, typical muscle-associat-ed proteins assessed by proteomics
Figure 3: Overview of groups of muscle-associated proteins that are routinely assessed by mass spectrometry-based proteomics.
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For citation purposes: Ohlendieck K. Proteomics of exercise-induced skeletal muscle adaptations. OA Sports Medicine 2013 Mar 01;1(1):3.
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in response to treadmill endurance overtraining using 2D gel electro-phoresis23, rat gastrocnemius muscle after one bout of an exhaustive exer-cise using 2D gel electrophoresis24, rat tibialis anterior and soleus muscle protein carbonylation in response to training25, rat epitrochlearis muscle in response to high intensity swim-ming using 2D-DIGE analysis26, horse vastus lateralis muscle following different stages of endurance train-ing using 2D gel electrophoresis27 and mouse leg muscles with insulin-like growth factor- mediated gene doping in response to endurance training28.
lateralis muscle in response to down-hill running-induced muscle damage using the 2D DIGE method18.
In animal studies, the effect of en-hanced neuromuscular activity was evaluated with rat plantaris muscle in response to moderate intensity endurance training using 2D gel elec-trophoresis19, rabbit tibialis anterior muscle following 14 and 60 days of chronic low-frequency stimulation using 2D gel electrophoresis20 and 2D-DIGE analysis21, rat gastrocnemius muscle in response to high intensity swimming using 2D gel electropho-resis22, rat gastrocnemius muscle
in response to 14 consecutive days of endurance training using the 2D-DIGE technique14, human soleus and vastus lateralis muscle in response to vibration exercise countermeas-ures to prevent muscular atrophy in lower limbs due to long-term bed rest using 2D-DIGE analysis15, the secretome of human skeletal muscle cells from vastus lateralis and trape-zius muscle in response to strength training using Nano-LC-LTQ Orbit-rap-MS/MS analysis16, human rectus femoris muscle in response to acute or repeated eccentric exercises using 2D-DIGE analysis17 and human vastus
Figure 4: Flowchart of the proteomic identification of a contractile protein from human vastus lateralis muscle. The scheme highlights the various steps in the unequivocal identification of the slow isoform of troponin TnT. The comparison of mass spectrometrically determined peptide sequences with a proteomic databank and subsequent protein identification is presented.
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For citation purposes: Ohlendieck K. Proteomics of exercise-induced skeletal muscle adaptations. OA Sports Medicine 2013 Mar 01;1(1):3.
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in the expression profile of the mitochondrial proteome, especially affecting enzymes such as NADH de-hydrogenase and ATP synthase14. The mitochondria-enriched fraction from skeletal muscle biopsies showed dif-ferential expression patterns of en-zymes of the citric acid cycle, oxidative phosphorylation, mitochondrial pro-tein synthesis, oxygen transportation and antioxidant capacity following endurance training14.
increasing fatigue resistance and modifying metabolic processes to maximize aerobic capacity30. The physiological conditioning of fatigue resistance and the bio-energetic en-hancement of aerobic performance are based on finely tuned physi-ological machinery that promotes endurance performance31. Proteomic profiling of the effect of endurance training on human vastus lateralis muscle revealed distinct adaptations
Proteomics of endurance exerciseThe neuromuscular system has an enormous capacity to adapt to a great variety of physical demands and differing training conditions by muscle remodelling involving chang-es in contractile properties, meta-bolic pathways and tissue mass29. In this respect, human skeletal muscles exhibit an extraordinary capacity to adjust to long-lasting endurance ex-ercise by optimizing power output,
Table 1 Proteomic changes in skeletal muscles in response to physical exerciseProteomic profiling studies
Skeletal muscle type and species
Methodological approach Protein changes References
Interval training Human vastus lateralis 2D-GE, iTRAQ ATP synthase, SDH, PTM of TnT, PTM of CK Holoway et al.13
Endurance training Human vastus lateralis 2D-DIGE
Adaptive response of mito-chondria; GYL-E to OxPhos-
E/CAC-E shift (NADH-DH, ATP synthase)
Egan et al.14
Vibration exercise during long-term bed rest
Human soleus and vastus lateralis
2D-DIGE MHC, OxPhos-E, CAC-E Moriggi et al.15
Strength training Human skeletal muscle cells
Nano-LC-MS/MS analysis
Release of various myokines (muscle secretome) Norheim et al.16
Repeated eccentric exercises Human rectus femoris 2D-DIGE MHC, GLY-E Hody et al.17
Downhill running- induced muscle damage Human vastus lateralis 2D-DIGE ACT, DES, CSQ Malm and Yu18
Moderate intensity endurance training Rat plantaris 2D-GE MYO, MLC2, GLY-E Burniston19
Chronic low-frequency electro-stimulation Rabbit tibialis anterior 2D-GE, 2D-DIGE
MYO, FABP3, CK, MHC, MLC, Tn, Tp, SERCA, RyR-
CRC, DHPR, CSQ
Donoghue et al.20,21
High intensity swimming Rat gastrocnemius 2D-GE TnT, CK Guelfi et al.22
Treadmill endurance overtraining Rat gastrocnemius 2D-GE MHC, OxPhos-E, CAC-E Gandra et al.23
One bout of an exhaustive exercise Rat gastrocnemius 2D-GE GLY-E, OxPhos-E Gandra et al.24
Training Rat tibialis anterior and soleus
GLY-E, NADH-DH, PTM changes in various muscle proteins (carbonylation)
Magherini et al.25
High intensity swimming Rat epitrochlearis 2D-DIGE Mitochondrial enzymes (NADH-DH), PVA
Yamaguchi et al.26
Endurance training Horse vastus lateralis 2D-GE ACT, GLY-E Bouwman et al.27
Endurance training following gene doping Various mouse leg muscles 2D-GE GLY-E-to-OxPhos-E/CAC-E
shift Macedo et al.28
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and lists the highly complex cellular processes that are involved in mus-cle fibre transitions, such as various degrees of fibre transformation, hypertrophy, neogenesis, atrophy, apoptosis and necrosis.
Proteomics of high-intensity trainingThe global effects of high-intensity training during strenuous interval training or strength training have also been analysed by proteomics13,16,22,24,26. Human vastus lateralis muscle showed increased expression levels of the mitochondrial enzymes succinate dehydrogenase and ATP synthase in response to interval training, as well as post-translational modulations of troponin TnT and muscle creatine ki-nase13. Similar results were obtained with a rat model of high intensity exer-cise using swimming boats while car-rying a weight26 or treadmill training with incremental increases in speed until exhaustion24. The proteomic profiling of exercised rat epitrochle-aris muscle revealed elevated levels of mitochondrial enzymes, especially NADH dehydrogenase. In contrast, the cytosolic Ca2+-binding protein parvalbumin was reduced follow-ing high intensity exercise26. Changes in these muscle-associated proteins appear to represent distinct altera-tions in the fibre proteome following the stimulation of the AMP-activated protein kinase AMPK and elevation of sarcoplasmic Ca2+-levels during muscle contraction. Norheim and co-workers16 have initiated the pro-teomic identification of potential al-terations in the secretion of signalling proteins from human skeletal muscle cells in response to strength training. Initial studies suggest that several types of myokines with paracrine or endocrine functions may be synthe-sized in myofibres and then being secreted for interactions with other tissues16. The exact activation process, release mechanisms and non-muscle targets of these novel protein factors remain to be determined.
countermeasure to prevent severe complications due to muscular atro-phy by proteomics15,20,21. The large-scale analysis of an established model for microgravity, which is presented by 8 weeks of horizontal bed rest, confirmed structural, functional and metabolic alterations in response to muscular disuse15. Altered distribu-tion patterns of myosin heavy chain isoforms and the lower abundance of enzymes involved in aerobic me-tabolism established increased type I fibres and decreased type IIA fibres in human soleus and vastus lateralis muscle in response to long-term bed rest. Resistive vibration exercise was shown to partially reverse these disuse-associated protein changes in lower limbs15. Newly recognized muscle proteins that change during extended periods of horizontal bed rest can now be further character-ized and tested for possible inclusion in the biomarker signature of reha-bilitation.
Chronic external stimulation of muscles has been applied in inno-vative medical applications such as the prevention of progressive mus-cle wasting in comatose patients or as cardiac assist devices in dynamic cardiomyoplasty. Proteomic analyses were carried out with an established animal model of stimulation-induced muscle transformation, the chronic low-frequency stimulated rabbit tibialis anterior muscle20,21. Chronic stimulation at a frequency of 10 Hz caused swift fast-to-slow transitions in isoforms of myosins, troponins and tropomyosins, as well as Ca2+-regula-tory pumps, channels and binding proteins. Changes in metabolic en-zymes indicated a glycolytic-to-oxi-dative shift in a slower-contracting fibre population20,21. These prot-eomic findings suggest that chronic electro-stimulation therapy is an ex-cellent option as a countermeasure to pathophysiological unloading of muscles. Figure 5 summarizes the effects of increased neuromuscu-lar activity on key muscle proteins
A variety of proteomic surveys with animal models of endurance training have confirmed exercise-induced mitochondrial remodelling and an increased capacity for oxidative me-tabolism. A clear bioenergetic shift from glycolysis towards fatty acid oxidation exists in several trained animal species19,25,27. Thus, the fact that endurance exercise results in mitochondrial remodelling and an increased oxidative capacity, rather than hypertrophy of muscle fibres, was confirmed by mass spectrome-try-based proteomics. Interestingly, adenovirus-mediated delivery of cDNA encoding insulin-like growth factor-I triggered neovascularization, muscular hypertrophy, fast-to-slow muscle transformation and a consid-erable endurance gain28. This shows the crucial role of growth factors in metabolic and functional adaptations of the neuromuscular system dur-ing training. However, in the case of excessive physical exercise, muscle fibres might be challenged by the lack of a sufficient supply of oxygen. This makes the findings of proteomic analyses of muscle fibres under con-ditions of hypoxia relevant for sports medicine. Chronic hypoxia triggers functional adaptations in skeletal muscles and causes a metabolically compensatory enhancement of the glycolytic pathway to counteract the lack of oxygen32.
Proteomics of vibration exercise and electro-stimulation therapyMuscular atrophy is a severe patho-physiological consequence of a variety of conditions involving neuromuscular unloading, such as motor neuron disease, traumatic denervation, limb immobilization, long-term bed rest in seriously ill patients, muscular disuse in coma-tose patients, exposure of astronauts to microgravity or the natural aging process. Over the last few years, the application of vibration exercise and chronic electro-stimulation therapy has been evaluated as a potential
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carbonic anhydrase, the application of proteomics promises to identify improved markers of rhabdomyoly-sis, as well as indicators of the natu-ral secretion process that releases myokines and other fibre-associated indicators during exercise-induced adaptations.
Proteomics has so far been applied to determine global changes in the case of delayed-onset muscle sore-ness in response to acute or repeat-ed eccentric exercises17 and skeletal muscle damage as a result of exten-sive downhill running18 in human
imbalances, disturbed electrolyte homeostasis, cardiac arrhythmia and acute kidney failure. In order to im-prove diagnostic methods to swiftly detect exercise-induced rhabdomy-olysis and be able to better evaluate the degree of skeletal muscle dam-age, new and more reliable fibre- derived biomarkers are needed. Mass spectrometry-based proteomics pre-sents an ideal analytical tool to estab-lish a superior biomarker signature of exercise-related muscle damage33. Besides the currently used serum biomarkers, creatine kinase and
Proteomics of overtraining and muscular injuryBesides neuromuscular diseases, traumatic injury, toxic insults, alco-hol abuse and pharmacological side effects, acute skeletal muscle damage can also be triggered by strenuous exercise. Vigorous strength training can put athletes at risk of severe fi-bre injury or even rhabdomyolysis. If muscle fibre breakdown triggers the extensive release of the intracel-lular muscle contents, the deleteri-ous leakage of fibre proteins and ions may cause pathological fluid
Figure 5: Schematic overview of the effects of increased neuromuscular activity on key muscle proteins (CSQ, calsequestrin; DHPR, dihydropyridine receptor; FABP, fatty acid binding protein; MHC, myosin heavy chain; MLC, myosin light chain; SERCA, sarcoplasmic reticulum Ca2+-ATPase). Listed are the various cellular processes that are believed to be involved in muscle fibre transitions, including fibre transformation, hypertrophy, neogenesis, atrophy, apoptosis and necrosis.
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For citation purposes: Ohlendieck K. Proteomics of exercise-induced skeletal muscle adaptations. OA Sports Medicine 2013 Mar 01;1(1):3.
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increased external loading via the IGF1/Akt/mTOR signalling path-way38, (iii) exercise-induced in-creases in mitochondrial biogenesis are mediated by PGC1-alpha39, (iv) epigenetic factors such as antisense RNA play a role in muscle gene reg-ulation40, (v) small non-coding mi-croRNAs are involved in the regu-lation of cellular proliferation and differentiation in skeletal muscles41, (vi) exercising skeletal muscle may act as an endocrine organ that pro-duces and releases hormone-like myokines and thereby exerts signal-ling effects on other organ systems in the body42, (vii) the dynamics of extra- and intramuscular connective tissue systems plays a central role in force transmission in skeletal mus-cle43, (viii) myonuclear addition is required during skeletal muscle hypertrophy44, (ix) Pax7-positive satellite cells are essential in acute injury-induced skeletal muscle re-generation45 and (x) certain geno-types correlate with phenotypes of enhanced endurance or power per-formance46. Building on these key findings, the next step in sports med-icine will be a systematic in-depth analysis of changes in the neuromus-cular system in response to exercise, which combines genome-, proteome- and metabolome-wide analyses.
ConclusionRapid advancements in protein bio-chemical techniques and the stream-lining of mass spectrometry-based proteomic workflows have enabled the establishment of global altera-tions in the concentration, isoform expression patterns, molecular in-teractions and post-translational modifications of muscle proteins following physical exercise. The sys-tematic application of proteomics has identified adaptive changes to training in key proteins involved in excitation–contraction coupling, the contraction–relaxation cycle, meta-bolic pathways and the cellular stress response. These findings have both
moderate endurance training a suit-able intervention to prevent cardiac failure34. The proteomic profiling of the rectus abdominus muscle from obese women has revealed a com-pensatory glycolytic drift probably to counteract reduced muscle mito-chondrial function during the pro-gression of obesity35. It will be of interest to investigate whether exer-cise can reverse this obesity-related metabolic syndrome and increase oxidative capacity to levels as nor-mally seen in healthy lean muscle tissue. A large number of proteomic studies have studied the effects of type 2 diabetes and metabolic im-pairments on the muscle proteome36. Insulin-resistant human muscle was demonstrated to be associated with an oxidative-to-glycolytic shift. Regu-lar exercise and a change in life style can be used to reverse this metabolic disturbance and thus counteract the negative effects of abnormal in-sulin signalling in diabetes and the metabolic syndrome. Figure 6 sum-marizes the pathological impact of genetic muscle diseases, insulin re-sistance and common co-morbidities on skeletal muscles and how changes in nutrition, enhanced physical ex-ercise levels and certain therapeutic interventions can be used for skeletal muscle regeneration. In the future, proteomics will be instrumental to identify novel biomarkers for the evaluation of the beneficial aspects of physical exercise and its preventative and clinical applications.
Recent advances in exercise biologyNew concepts in exercise biology are highlighted by discoveries that have demonstrated that (i) novel signalling molecules of the cellular energy status are majorly engaged in skeletal muscle metabolism, such as the AMP-activated protein kinase and its activating role in glucose dis-posal and fatty acid oxidation37, (ii) mechanical stimuli regulate mus-cle fibre size under conditions of
athletes, as well as in an animal model of overtraining using an exces-sive treadmill endurance exercise23. Surprisingly, myosin heavy chains and glycolytic enzymes decreased after eccentric tests, suggesting that eccentric training may trigger a switch to oxidative metabolism to protect against delayed-onset mus-cle soreness17. Downhill running-associated skeletal muscle damage was found to induce increased levels of actin and desmin, but a reduction in the luminal Ca2+-binding protein calsequestrin of the sarcoplasmic reticulum. Hence, cytoskeletal func-tions, the assembly and stabilization of the Z-disc domain and calcium homeostasis seem to be affected in running-related muscle damage18. The proteomic profiling of different parts of rat gastrocnemius muscle has shown that skeletal muscles with different fibre-type compositions respond differently in response to treadmill endurance overtraining23. The red portion of the over-trained gastrocnemius muscle exhibited an increased density of proteins in-volved in oxidative phosphorylation, lipid metabolism, antioxidant pro-tection and the cellular stress re-sponse23, as usually observed during adaptations to endurance training. Interestingly, the white portion of the same muscle did not show these al-terations following treadmill endur-ance overtraining23.
Muscle proteomics and preventative medicineVarious common diseases are di-rectly or indirectly associated with muscle weakness, fibre degenera-tion or abnormal muscle metabo-lism. This includes type 2 diabetes, obesity, heart failure, kidney disease, chronic obstructive pulmonary dis-ease and cancer cachexia, and also the natural aging process. Regular physical exercise and a balanced diet were clearly shown to have ben-eficial effects on cardiac and skeletal muscle energy metabolism, making
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For citation purposes: Ohlendieck K. Proteomics of exercise-induced skeletal muscle adaptations. OA Sports Medicine 2013 Mar 01;1(1):3.
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Abbreviations2D, two-dimensional; ACT, actin; CAC-E, citric acid cycle enzymes; CSQ, calsequestrin; CK, creatine kinase; DES, desmin; DHPR, dihy-dropyridine receptor; DIGE, dif-ference in-gel electrophoresis; DH, dehydrogenase; FABP, fatty acid binding protein; GE, gel electropho-resis; GLY-E, glycolytic enzymes;
of future proteomic and systems bio-logical studies in sports medicine.
AcknowledgementsResearch in the author’s laboratory was supported by project grants from Muscular Dystrophy Ireland and the BioAT programme of PRTLI cycle 5 of the Irish Higher Education Authority.
improved our general understanding of molecular and cellular mecha-nisms that underlie skeletal muscle transitions and identified interesting new biomarker candidates that are characteristic for exercise-induced muscle transformation. Recent phys-iological, biochemical and genetic advances in the field of exercise sci-ence will heavily influence the design
Figure 6: Flowchart summarizing the pathological impact of genetic muscle diseases, insulin resistance and common co-morbidities on skeletal muscles. The scheme highlights how changes in life style and enhanced physical exercise levels can play a crucial role in therapeutic interventions to promote skeletal muscle regeneration.
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For citation purposes: Ohlendieck K. Proteomics of exercise-induced skeletal muscle adaptations. OA Sports Medicine 2013 Mar 01;1(1):3.
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exercise on skeletal muscle proteins in fasted rats. Clin Exp Pharmacol Physiol. 2006 Oct;33(10):952–7.23. Gandra PG, Valente RH, Perales J,Pacheco AG, Macedo DV. Proteomic pro-filing of skeletal muscle in an animal model of overtraining. Proteomics. 2012 Aug;12(17):2663–7.24. Gandra PG, Valente RH, Perales J, Pa-checo AG, Macedo DV. Proteomic analysis of rat skeletal muscle submitted to one bout of incremental exercise. Scand J Med Sci Sports. 2012 Apr;22(2):207–16.25. Magherini F, Abruzzo PM, Puglia M,Bini L, Gamberi T, Esposito F, et al. Pro-teomic analysis and protein carbonyla-tion profile in trained and untrained rat muscles. J Proteomics. 2012 Jan;75(3): 978–92.26. Yamaguchi W, Fujimoto E, HiguchiM, Tabata I. A DIGE proteomic analy-sis for high-intensity exercise-trained rat skeletal muscle. J Biochem. 2010 Sep;148(3):327–33.27. Bouwman FG, van Ginneken MM, No-ben JP, Royackers E, de Graaf-Roelfsema E, Wijnberg ID, et al. Differential expres-sion of equine muscle biopsy proteins during normal training and intensified training in young standard bred horses using proteomics technology. Comp Bio-chem Physiol Part D Genom Proteom. 2010 Mar;5(1):55–64.28. Macedo A, Moriggi M, Vasso M, DePalma S, Sturnega M, Friso G, et al. En-hanced athletic performance on multi-site AAV-IGF1 gene transfer coincides with massive modification of the mus-cle proteome. Hum Gene Ther. 2012 Feb;23(2):146–57.29. Bassel-Duby R, Olson EN. Signalingpathways in skeletal muscle remodeling. Annu Rev Biochem. 2006;75:19–37.30. Flueck M, Eilers W. Training modali-ties: impact on endurance capacity. En-docrinol Metab Clin North Am. 2010 Mar;39(1):183–20031. Seene T, Kaasik P, Alev K. Muscleprotein turnover in endurance train-ing: a review. Int J Sports Med. 2011 Dec;32(12):905–11.32. Viganò A, Ripamonti M, De Palma S,Capitanio D, Vasso M, Wait R, et al. Pro-teins modulation in human skeletal muscle in the early phase of adaptation to hypobaric hypoxia. Proteomics. 2008 Nov;8(22):4668–79.33. Ohlendieck K. Proteomic iden-tification of biomarkers of skeletal
12. Ohlendieck K. Proteomics of skeletalmuscle differentiation, neuromuscular disorders and fibre aging. Expert Rev Proteomics. 2010 Apr;7(2):283–96.13. Holloway KV, O'Gorman M, WoodsP, Morton JP, Evans L, Cable NT, et al. Proteomic investigation of changes in hu-man vastus lateralis muscle in response to interval-exercise training. Proteomics. 2009 Nov;9(22):5155–74.14. Egan B, Dowling P, O'Connor PL,Henry M, Meleady P, Zierath JR, et al. 2-D DIGE analysis of the mitochondrial proteome from human skeletal muscle reveals time course-dependent remodel-ling in response to 14 consecutive days of endurance exercise training. Proteomics. 2011 Apr;11(8):1413–28.15. Moriggi M, Vasso M, Fania C, Capitanio D, Bonifacio G, Salanova M, et al. Long term bed rest with and without vibration exercise countermeasures: effects on hu-man muscle protein dysregulation. Prot-eomics. 2010 Nov;10(21):3756–74.16. Norheim F, Raastad T, Thiede B,Rustan AC, Drevon CA, Haugen F. Prot-eomic identification of secreted proteins from human skeletal muscle cells and expression in response to strength train-ing. Am J Physiol Endocrinol Metab. 2011 Nov;301(5):E1013–21.17. Hody S, Leprince P, Sergeant K, Renaut J, Croisier JL, Wang F, et al. Human mus-cle proteome modifications after acute or repeated eccentric exercises. Med Sci Sports Exerc. 2011 Dec;43(12):2281–96.18. Malm C, Yu JG. Exercise-induced mus-cle damage and inflammation: re-evalua-tion by proteomics. Histochem Cell Biol. 2012 Jul;138(1):89–99.19. Burniston JG. Changes in the ratskeletal muscle proteome induced by moderate-intensity endurance exercise. Biochim Biophys Acta. 2008 Jul;1784 (7-8):1077–86.20. Donoghue P, Doran P, Dowling P,Ohlendieck K. Differential expression of the fast skeletal muscle proteome fol-lowing chronic low-frequency stimu-lation. Biochim Biophys Acta. 2005 Sep;1752(2):166–76.21. Donoghue P, Doran P, Wynne K, Ped-ersen K, Dunn MJ, Ohlendieck K. Prot-eomic profiling of chronic low-frequency stimulated fast muscle. Proteomics. 2007 Sep;7(18):3417–30.22. Guelfi KJ, Casey TM, Giles JJ, Fourni-er PA, Arthur PG. A proteomic analysis of the acute effects of high-intensity
iTRAQ, isobaric tags for relative and absolute quantitation; MHC, myosin heavy chain; MLC, myo-sin light chain; MYO, myoglobin; OxPhos-E, oxidative phosphoryla-tion enzymes; PVA, parvalbumin; RyR-CRC, ryanodine receptor Ca2+-release channel; SDH, succinate de-hydrogenase; SERCA, sarcoplasmic reticulum Ca2+-ATPase; Tn, troponin (T, I, C); Tp, tropomyosin; PTM, post-translational modifications.
References1. Baldwin KM, Haddad F. Research inthe exercise sciences: where we are and where do we go from here—Part II. Exerc Sport Sci Rev. 2010 Apr;38(2):42–50.2. Harridge SD. Plasticity of humanskeletal muscle: gene expression to in vivo function. Exp Physiol. 2007 Sep;92(5):783–97.3. Flück M. Functional, structural and mo-lecular plasticity of mammalian skeletal muscle in response to exercise stimuli. J Exp Biol. 2006 Jun;209(12):2239–48.4. Booth FW, Laye MJ. The future: genes,physical activity and health. Acta Physiol (Oxf). 2010 Aug;199(4):549–56.5. Hittel DS, Hathout Y, Hoffman EP. Pro-teomics and systems biology in exercise and sport sciences research. Exerc Sport Sci Rev. 2007 Jan;35(1):5–11.6. Burniston JG, Hoffman EP. Proteomicresponses of skeletal and cardiac muscle to exercise. Expert Rev Proteomics. 2011 Jun;8(3):361–77.7. Petriz BA, Gomes CP, Rocha LA,Rezende TM, Franco OL. Proteomics ap-plied to exercise physiology: A cutting-edge technology. J Cell Physiol. 2012 Mar;227(3):885–98.8. Paik YK, Jeong SK, Omenn GS, Uhlen M,Hanash S, Cho SY, et al. The Chromosome-Centric Human Proteome Project for cat-aloging proteins encoded in the genome. Nat Biotechnol. 2012 Mar;30(3):221–3.9. Altelaar AF, Munoz J, Heck AJ. Next-generation proteomics: towards an inte-grative view of proteome dynamics. Nat Rev Genet. 2013 Jan;14(1):35–48.10. Ohlendieck K. Skeletal muscle pro-teomics: Current approaches, technical challenges and emerging techniques. Skelet Muscle. 2011 Feb 1;1(1):6.11. Lewis C, Doran P, Ohlendieck K. Pro-teomic analysis of dystrophic muscle. Methods Mol Biol. 2012;798:357–69.
Page 12 of 12
Critical review
Licensee OA Publishing London 2013. Creative Commons Attribution License (CC-BY)
For citation purposes: Ohlendieck K. Proteomics of exercise-induced skeletal muscle adaptations. OA Sports Medicine 2013 Mar 01;1(1):3.
Com
petin
g in
tere
sts:
non
e de
clar
ed. C
onfli
ct o
f int
eres
ts: n
one
decl
ared
. A
ll au
thor
s co
ntrib
uted
to c
once
ption
and
des
ign,
man
uscr
ipt p
repa
ratio
n, re
ad a
nd a
ppro
ved
the
final
man
uscr
ipt.
All
auth
ors
abid
e by
the
Ass
ocia
tion
for M
edic
al E
thic
s (A
ME)
eth
ical
rule
s of
dis
clos
ure.
43. Mackey AL, Heinemeier KM, Koski-nen SO, Kjaer M. Dynamic adaptation of tendon and muscle connective tissue to mechanical loading. Connect Tissue Res. 2008;49(3):165–8.44. Petrella JK, Kim JS, Mayhew DL, CrossJM, Bamman MM. Potent myofiber hy-pertrophy during resistance training in humans is associated with satellite cell-mediated myonuclear addition: a cluster analysis. J Appl Physiol. 2008 Jun;104(6):1736–42.45. Lepper C, Partridge TA, Fan CM. Anabsolute requirement for Pax7-positive satellite cells in acute injury-induced skeletal muscle regeneration. Develop-ment. 2011 Sep;138(17):3639–46.46. Roth SM, Rankinen T, Hagberg JM,Loos RJ, Pérusse L, Sarzynski MA, et al. Advances in exercise, fitness, and perfor-mance genomics in 2011. Med Sci Sports Exerc. 2012 May;44(5):809–17.
in skeletal muscle. Int J Biochem Cell Biol. 2011 Sep;43(9):1267–76.39. Hood DA. Mechanisms of exercise-induced mitochondrial biogenesis in skeletal muscle. Appl Physiol Nutr Metab. 2009 Jun;34(3):465–72.40. Pandorf CE, Haddad F, Roy RR, QinAX, Edgerton VR, Baldwin KM. Dynamics of myosin heavy chain gene regulation in slow skeletal muscle: role of natural antisense RNA. J Biol Chem. 2006 Dec 15;281(50):38330–42.41. Townley-Tilson WH, Callis TE, WangD. MicroRNAs 1, 133, and 206: criti-cal factors of skeletal and cardiac mus-cle development, function, and dis-ease. Int J Biochem Cell Biol. 2010 Aug; 42(8):1252–5.42. Pedersen BK, Febbraio MA. Muscles,exercise and obesity: skeletal muscle as a secretory organ. Nat Rev Endocrinol. 2012 Apr 3;8(8):457–65.
muscle disorders. Biomark Med. 2013 Feb;7(1):169–86.34. Ventura-Clapier R, Mettauer B, Bigard X. Beneficial effects of endurance training on cardiac and skeletal muscle energy metabolism in heart failure. Cardiovasc Res. 2007 Jan;73(1):10–8.35. Hittel DS, Hathout Y, Hoffman EP,Houmard JA. Proteome analysis of skel-etal muscle from obese and morbidly obese women. Diabetes. 2005 May;54(5): 1283–8.36. Ohlendieck K. Pathobiochemicalchanges in diabetic skeletal muscle as re-vealed by mass-spectrometry-based prot-eomics. J Nutr Metab. 2012;2012:893876.37. Winder WW, Thomson DM. Cellularenergy sensing and signaling by AMP-activated protein kinase. Cell Biochem Biophys. 2007;47(3):332–47.38. Hornberger TA. Mechanotransduction and the regulation of mTORC1 signaling
