Proteomics of exercise-induced skeletal muscle adaptations

Muscle Biology Laboratory, Department of Biology, National University of Ireland, Maynooth, Co. Kildare, Ireland

* Corresponding author Email: kay.ohlendieck@nuim.ie

Introduction

The systems biological analysis of dynamic protein constellations and the determination of proteome-wide alterations due to physiological adaptations play an increasing role in modern sports medicine. Several large-scale studies on the effect of physical training in humans and relevant animal models have decisively improved our global understanding of the molecular and cellular mechanisms involved in skeletal muscle changes during exercise. The aim of this critical review was to discuss the proteomics of exercise-induced skeletal muscle adaptations.

Discussion

Building on this extensive knowledge of conventional exercise biology, refined protein biochemical and mass spectrometric technologies can now be employed to study subtle changes in protein concentration, isoform expression patterns, protein-protein interactions and/or post-translational modifications following physical activity. Besides being a key method for the elucidation of fibre plasticity and muscle transformation, the systematic application of mass spectrometry-based proteomics promises to play a prevalent role in the establishment and evaluation of preventative exercise regimes to counteract skeletal muscle wasting and metabolic disturbances in common disorders with muscular involvement such as diabetes, obesity, cardiovascular disease, cancer cachexia or sarcopenia of old age. In this critical review, the impact of recent proteomic profiling studies of physical exercise is examined and its implications for our molecular understanding of skeletal muscle adaptations are discussed.

Conclusion

Recent findings from mass spectrometry-based proteomic studies of physical exercise have identified a variety of adaptive changes in muscle proteins involved in cellular signalling, fibre contraction, metabolic pathways and the cellular stress response. The establishment of these novel biomarkers, which are characteristic for exercise-related muscle adaptations, will be extremely useful for the detailed biochemical evaluation of physical training programs.

Over the last decade, a large number of scientific breakthroughs have transformed the field of exercise biology^[1]. Our understanding of gene regulation and protein alterations in response to physical exercise has dramatically improved through the application of molecular and cellular analyses of skeletal muscle adaptations. This has involved the elucidation of novel structural, functional and metabolic aspects during force generation and physiological adaptability in response to different training regimes^[2]. Exercise triggers diverse physiological stimuli that involve neuronal, mechanical, metabolic and hormonal signals that are sensed, transduced and integrated in a highly coordinated manner^[3]. The repeated recruitment of specific muscle groups causes lasting alterations in gene expression patterns and distinct changes in the concentration, isoform repertoire and/or post-translational modifications of skeletal muscle proteins^[4].

During muscle adaptations, a crucial relationship exists between contraction-induced signalling cascades 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-induced physiological disturbances on the level of the genome, transcriptome, proteome and metabolome^[5,6,7]. 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 rationalize the physiological and biochemical basis of muscle adaptations and to suitably plan future global studies in exercise biology.

The author has referenced some of his own studies in this review. These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed. All human subjects, in these referenced studies, gave informed consent to participate in these studies.

From genome to muscle proteome

Following the elucidation of the human genome and a variety of animal model genomes of physiological or pathological relevance, a major challenge in the field of biomedicine is now presented by the determination of the cell-specific activation of all identified genes and the many functions of their expressed protein products. The biochemical characterization of the proteins encoded by the approximately 20,300 human genes is complicated by the multi-functionality of many protein molecules, the highly diverse interactions within protein complexes and the dynamic nature of protein expression patterns^[8]. 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 environmental influences. The discrepancy between the extremely large number 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 cleavage of some polypeptide chains into more than one subunit, as well as an extremely large variety of post-translational modifications^[9].

The ultimate goal of applying a systems biological approach to the field of applied myology is the establishment of a unifying scheme that explains how the metabolic status and a plethora of physiological stimuli from extracellular and intramuscular systems result in functional and structural adaptations to enhanced neuromuscular activity^[4]. Figure 1 outlines the relationship between muscle activity-induced signalling, the integration of these physiological 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 systems, its functional behaviour during an altered physiological state may be useful for establishing the molecular mechanisms that underlie the plasticity of the physiome. A crucial part of the physiological dynamics of the neuromuscular system is based on alterations in protein density, protein localization, protein isoform expression, protein-protein interactions and post-translational modifications.

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.

Proteomic profiling of muscle biopsies

In order to study the accessible proteome from skeletal muscle tissues, highly efficient methods for extraction, fractionation, separation and detection of proteins have to be combined in a rationalized workflow^[10]. Figure 2 summarizes the main steps involved in gel electrophoresis-based proteomic studies of muscle adaptations during exercise. Alternatively, proteins can be separated by liquid chromatography or a combination of chromatographical and electrophoretic methodology. Following the preparation of total protein extracts or the separation of individual organelles, components from varying proteomes can be differentially labelled with fluorescent dyes prior to gel electrophoresis. The fluorescence difference in-gel electrophoretic (DIGE) method can visualize several thousand muscle proteins in a single analytical experiment, making it an extremely valuable tool for comparative proteomics^[11]. Fluorescently tagged proteins are routinely separated by high-resolution two-dimensional gel electrophoresis using isoelectric focusing in the first dimension and sodium dodecyl sulphate polyacrylamide slab gel electrophoresis in the second dimension. Following the densitometric analysis of spot patterns, proteins of interest are excised and digested for mass spectrometric peptide analysis. Peptide sequences are compared to international databanks for the unequivocal identification of individual protein species.

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).

The differential expression of identified protein candidates is then usually verified by immunoblotting surveys. The biochemical, cell biological and physiological characterization of novel proteins is routinely carried out by enzyme assays, binding tests, confocal microscopy and functional analyses^[10]. As summarized in Figure 3, typical muscle-associated proteins assessed by proteomics include elements involved in neuromuscular transmission, excitation-contraction coupling, ion homeostasis, signal transduction, fibre assembly, contraction, relaxation, fibre elasticity, cytoskeletal maintenance, metabolic integration, metabolite transportation, glycolysis, fatty acid oxidation, citric acid cycle, oxidative phosphorylation, lipid metabolism, nucleotide metabolism, gene regulation, transcription, translation, protein synthesis, protein assembly, protein storage, fibre repair, neogenesis, immune response, detoxification and the cellular stress response^[12]. Figure 4 highlights the various steps in the proteomic identification of a key protein of the contractile 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 generated peptide population is analysed by mass spectrometry and the amino acid sequence of individual peptides compared to databanks containing sequence information of the human proteome. The peptide information resulted in a 20% sequence coverage, 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.

Figure 3: Overview of groups of muscle-associated proteins that are routinely assessed by mass spectrometry-based proteomics.

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.

Exercise proteomics

In the field of sports medicine, the application of mass spectrometry-based proteomics attempts to identify global mechanisms of protein alterations that support the establishment of the endurance phenotype or power performance^[5,6,7]. Exercise proteomics was used to study protein alterations in humans^{[13,14,15,16,17,18]} and animal models of physical activity^{[19,20,21,22,23,24,25,26,27,28]}, as summarized in Table 1. This has included the analysis of human vastus lateralis muscle in response to interval training using both 2D gel electrophoresis and the quantitative isobaric tags for relative and absolute quantitation (iTRAQ) method^[13], the mitochondrial proteome from human vastus lateralis muscle in response to 14 consecutive days of endurance training using the 2D-DIGE technique^[14], human soleus and vastus lateralis muscle in response to vibration exercise countermeasures to prevent muscular atrophy in lower limbs due to long-term bed rest using 2D-DIGE analysis^[15], the secretome of human skeletal muscle cells from vastus lateralis and trapezius muscle in response to strength training using Nano-LC-LTQ Orbitrap-MS/MS analysis^[16], human rectus femoris muscle in response to acute or repeated eccentric exercises using 2D-DIGE analysis^[17] and human vastus lateralis muscle in response to downhill running-induced muscle damage using the 2D DIGE method^[18].

Table 1 Proteomic changes in skeletal muscles in response to physical exercise

In animal studies, the effect of enhanced neuromuscular activity was evaluated with rat plantaris muscle in response to moderate intensity endurance training using 2D gel electrophoresis^[19], rabbit tibialis anterior muscle following 14 and 60 days of chronic low-frequency stimulation using 2D gel electrophoresis^[20] and 2D-DIGE analysis^[21], rat gastrocnemius muscle in response to high intensity swimming using 2D gel electrophoresis^[22], rat gastrocnemius muscle in response to treadmill endurance overtraining using 2D gel electrophoresis^[23], rat gastrocnemius muscle after one bout of an exhaustive exercise using 2D gel electrophoresis^[24], rat tibialis anterior and soleus muscle protein carbonylation in response to training^[25], rat epitrochlearis muscle in response to high intensity swimming using 2D-DIGE analysis^[26], horse vastus lateralis muscle following different stages of endurance training using 2D gel electrophoresis^[27] and mouse leg muscles with insulin-like growth factor-mediated gene doping in response to endurance training^[28].

Proteomics of endurance exercise

The neuromuscular system has an enormous capacity to adapt to a great variety of physical demands and differing training conditions by muscle remodelling involving changes in contractile properties, metabolic pathways and tissue mass^[29]. In this respect, human skeletal muscles exhibit an extraordinary capacity to adjust to long-lasting endurance exercise by optimizing power output, increasing fatigue resistance and modifying metabolic processes to maximize aerobic capacity^[30]. The physiological conditioning of fatigue resistance and the bio-energetic enhancement of aerobic performance are based on finely tuned physiological machinery that promotes endurance performance^[31]. Proteomic profiling of the effect of endurance training on human vastus lateralis muscle revealed distinct adaptations in the expression profile of the mitochondrial proteome, especially affecting enzymes such as NADH dehydrogenase and ATP synthase^[14]. The mitochondria-enriched fraction from skeletal muscle biopsies showed differential expression patterns of enzymes of the citric acid cycle, oxidative phosphorylation, mitochondrial protein synthesis, oxygen transportation and antioxidant capacity following endurance training^[14].

A variety of proteomic surveys with animal models of endurance training have confirmed exercise-induced mitochondrial remodelling and an increased capacity for oxidative metabolism. A clear bioenergetic shift from glycolysis towards fatty acid oxidation exists in several trained animal species^[19,25,27]. 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 spectrometry-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 considerable endurance gain^[28]. This shows the crucial role of growth factors in metabolic and functional adaptations of the neuromuscular system during 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 conditions 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 oxygen^[32].

Proteomics of vibration exercise and electro-stimulation therapy

Muscular atrophy is a severe pathophysiological 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 comatose 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 countermeasure to prevent severe complications due to muscular atrophy by proteomics^[15,20,21]. 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 disuse^[15]. Altered distribution patterns of myosin heavy chain isoforms and the lower abundance of enzymes involved in aerobic metabolism 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 limbs^[15]. Newly recognized muscle proteins that change during extended periods of horizontal bed rest can now be further characterized and tested for possible inclusion in the biomarker signature of rehabilitation.

Chronic external stimulation of muscles has been applied in innovative medical applications such as the prevention of progressive muscle 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 muscle^[20,21]. Chronic stimulation at a frequency of 10 Hz caused swift fast-to-slow transitions in isoforms of myosins, troponins and tropomyosins, as well as Ca^[2+]-regulatory pumps, channels and binding proteins. Changes in metabolic enzymes indicated a glycolytic-to-oxidative shift in a slower-contracting fibre population^[20,21]. These proteomic findings suggest that chronic electro-stimulation therapy is an excellent option as a countermeasure to pathophysiological unloading of muscles. Figure 5 summarizes the effects of increased neuromuscular activity on key muscle proteins and lists the highly complex cellular processes that are involved in muscle fibre transitions, such as various degrees of fibre transformation, hypertrophy, neogenesis, atrophy, apoptosis and necrosis.

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 Ca[2+]-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.

Proteomics of high-intensity training

The global effects of high-intensity training during strenuous interval training or strength training have also been analysed by proteomics^{[13,16,22,24,26]}. 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 kinase^[13]. Similar results were obtained with a rat model of high intensity exercise using swimming boats while carrying a weight^[26] or treadmill training with incremental increases in speed until exhaustion^[24]. The proteomic profiling of exercised rat epitrochlearis muscle revealed elevated levels of mitochondrial enzymes, especially NADH dehydrogenase. In contrast, the cytosolic Ca^[2+]-binding protein parvalbumin was reduced following high intensity exercise^[26]. Changes in these muscle-associated proteins appear to represent distinct alterations in the fibre proteome following the stimulation of the AMP-activated protein kinase AMPK and elevation of sarcoplasmic Ca^[2+]-levels during muscle contraction. Norheim and co-workers^[16] have initiated the proteomic identification of potential alterations 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 synthesized in myofibres and then being secreted for interactions with other tissues^[16]. The exact activation process, release mechanisms and non-muscle targets of these novel protein factors remain to be determined.

Proteomics of overtraining and muscular injury

Besides neuromuscular diseases, traumatic injury, toxic insults, alcohol 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 fibre injury or even rhabdomyolysis. If muscle fibre breakdown triggers the extensive release of the intracellular muscle contents, the deleterious leakage of fibre proteins and ions may cause pathological fluid imbalances, disturbed electrolyte homeostasis, cardiac arrhythmia and acute kidney failure. In order to improve diagnostic methods to swiftly detect exercise-induced rhabdomyolysis and be able to better evaluate the degree of skeletal muscle damage, new and more reliable fibre-derived biomarkers are needed. Mass spectrometry-based proteomics presents an ideal analytical tool to establish a superior biomarker signature of exercise-related muscle damage^[33]. Besides the currently used serum biomarkers, creatine kinase and carbonic anhydrase, the application of proteomics promises to identify improved markers of rhabdomyolysis, as well as indicators of the natural 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 soreness in response to acute or repeated eccentric exercises^[17] and skeletal muscle damage as a result of extensive downhill running^[18] in human athletes, as well as in an animal model of overtraining using an excessive treadmill endurance exercise^[23]. 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 muscle soreness^[17]. Downhill running-associated skeletal muscle damage was found to induce increased levels of actin and desmin, but a reduction in the luminal Ca^[2+]-binding protein calsequestrin of the sarcoplasmic reticulum. Hence, cytoskeletal functions, the assembly and stabilization of the Z-disc domain and calcium homeostasis seem to be affected in running-related muscle damage^[18]. 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 overtraining^[23]. The red portion of the over-trained gastrocnemius muscle exhibited an increased density of proteins involved in oxidative phosphorylation, lipid metabolism, antioxidant protection and the cellular stress response^[23], as usually observed during adaptations to endurance training. Interestingly, the white portion of the same muscle did not show these alterations following treadmill endurance overtraining^[23].

Muscle proteomics and preventative medicine

Various common diseases are directly or indirectly associated with muscle weakness, fibre degeneration or abnormal muscle metabolism. This includes type 2 diabetes, obesity, heart failure, kidney disease, chronic obstructive pulmonary disease and cancer cachexia, and also the natural aging process. Regular physical exercise and a balanced diet were clearly shown to have beneficial effects on cardiac and skeletal muscle energy metabolism, making moderate endurance training a suitable intervention to prevent cardiac failure^[34]. The proteomic profiling of the rectus abdominus muscle from obese women has revealed a compensatory glycolytic drift probably to counteract reduced muscle mitochondrial function during the progression of obesity^[35]. It will be of interest to investigate whether exercise can reverse this obesity-related metabolic syndrome and increase oxidative capacity to levels as normally seen in healthy lean muscle tissue. A large number of proteomic studies have studied the effects of type 2 diabetes and metabolic impairments on the muscle proteome^[36]. Insulin-resistant human muscle was demonstrated to be associated with an oxidative-to-glycolytic shift. Regular exercise and a change in life style can be used to reverse this metabolic disturbance and thus counteract the negative effects of abnormal insulin signalling in diabetes and the metabolic syndrome. Figure 6 summarizes the pathological impact of genetic muscle diseases, insulin resistance and common co-morbidities on skeletal muscles and how changes in nutrition, enhanced physical exercise 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.

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.

Recent advances in exercise biology

New 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 disposal and fatty acid oxidation^[37], (ii) mechanical stimuli regulate muscle fibre size under conditions of increased external loading via the IGF1/Akt/mTOR signalling pathway^[38], (iii) exercise-induced increases in mitochondrial biogenesis are mediated by PGC1-alpha^[39], (iv) epigenetic factors such as antisense RNA play a role in muscle gene regulation^[40], (v) small non-coding microRNAs are involved in the regulation of cellular proliferation and differentiation in skeletal muscles^[41], (vi) exercising skeletal muscle may act as an endocrine organ that produces and releases hormone-like myokines and thereby exerts signalling effects on other organ systems in the body^[42], (vii) the dynamics of extra- and intramuscular connective tissue systems plays a central role in force transmission in skeletal muscle^[43], (viii) myonuclear addition is required during skeletal muscle hypertrophy^[44], (ix) Pax7-positive satellite cells are essential in acute injury-induced skeletal muscle regeneration^[45] and (x) certain genotypes correlate with phenotypes of enhanced endurance or power performance^[46]. Building on these key findings, the next step in sports medicine will be a systematic in-depth analysis of changes in the neuromuscular system in response to exercise, which combines genome-, proteome- and metabolome-wide analyses.

Rapid advancements in protein biochemical techniques and the streamlining of mass spectrometry-based proteomic workflows have enabled the establishment of global alterations in the concentration, isoform expression patterns, molecular interactions and post-translational modifications of muscle proteins following physical exercise. The systematic application of proteomics has identified adaptive changes to training in key proteins involved in excitation-contraction coupling, the contraction-relaxation cycle, metabolic pathways and the cellular stress response. These findings have both improved our general understanding of molecular and cellular mechanisms that underlie skeletal muscle transitions and identified interesting new biomarker candidates that are characteristic for exercise-induced muscle transformation. Recent physiological, biochemical and genetic advances in the field of exercise science will heavily influence the design of future proteomic and systems biological studies in sports medicine.

2D, two-dimensional; ACT, actin; CAC-E, citric acid cycle enzymes; CSQ, calsequestrin; CK, creatine kinase; DES, desmin; DHPR, dihydropyridine receptor; DIGE, difference in-gel electrophoresis; DH, dehydrogenase; FABP, fatty acid binding protein; GE, gel electrophoresis; GLY-E, glycolytic enzymes; iTRAQ, isobaric tags for relative and absolute quantitation; MHC, myosin heavy chain; MLC, myosin light chain; MYO, myoglobin; OxPhos-E, oxidative phosphorylation enzymes; PVA, parvalbumin; RyR-CRC, ryanodine receptor Ca^[2+]-release channel; SDH, succinate dehydrogenase; SERCA, sarcoplasmic reticulum Ca^[2+]-ATPase; Tn, troponin (T, I, C); Tp, tropomyosin; PTM, post-translational modifications.

Research 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.

All authors contributed to conception and design, manuscript preparation, read and approved the final manuscript.

None declared.

None declared.

All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.