A Systematic Review and Meta-Analysis of Proteomics Literature on the Response of Human Skeletal Muscle to Obesity/Type 2 Diabetes Mellitus (T2DM) Versus Exercise Training.

We performed a systematic review and meta-analysis of proteomics literature that reports human skeletal muscle responses in the context of either pathological decline associated with obesity/T2DM and physiological adaptations to exercise training. Literature was collected from PubMed and DOAJ databases following PRISMA guidelines using the search terms 'proteom*', and 'skeletal muscle' combined with either 'obesity, insulin resistance, diabetes, impaired glucose tolerance' or 'exercise, training'. Eleven studies were included in the systematic review, and meta-analysis was performed on a sub-set (four studies) of the reviewed literature that reported the necessary primary data. The majority of proteins (n = 73) more abundant in the muscle of obese/T2DM individuals were unique to this group and not reported to be responsive to exercise training. The main response of skeletal muscle to exercise training was a greater abundance of proteins of the mitochondrial electron transport chain, tricarboxylic acid cycle and mitochondrial respiratory chain complex I assembly. In total, five proteins were less abundant in muscle of obese/T2DM individuals and were also reported to be more abundant in the muscle of endurance-trained individuals, suggesting one of the major mechanisms of exercise-induced protection against the deleterious effects of obesity/T2DM occurs at complex I of the electron transport chain

Exercise-responsive phosphoproteins in the heart.

Endurance exercise improves cardiac performance and affords protection against cardiovascular diseases but the signalling events that mediate these benefits are largely unexplored. Phosphorylation is a widely studied post-translational modification involved in intracellular signalling, and to discover novel phosphorylation events associated with exercise we have profiled the cardiac phosphoproteome response to a standardised exercise test to peak oxygen uptake (VO2peak). Male Wistar rats (346±18g) were assigned to 3 independent groups (n=6, in each) that were familiarised with running on a motorised treadmill within a metabolic chamber. Animals performed a graded exercise test and were killed either immediately (0h) after or 3h after terminating the test at a standardised physiological end point (i.e. peak oxygen uptake; VO2peak). Control rats were killed at a similar time of day to the exercised animals, to minimise possible circadian effects. Cardiac proteins were digested with trypsin and phosphopeptides were enriched by selective binding to titanium dioxide (TiO2). Phosphopeptides were analysed by liquid chromatography and high-resolution tandem mass spectrometry, and phosphopeptides were quantified by MS1 intensities and identified against the UniProt knowledgebase using MaxQuant (data are available via ProteomeXchange, ID PXD006646). The VO2peak of rats in the 0h and 3h groups was 66±5mlkg(-1)min(-1) and 69.8±5mlkg(-1)min(-1), respectively. Proteome profiling detected 1169 phosphopeptides and one-way ANOVA found 141 significant (P<0.05 with a false discovery rate of 10%) differences. Almost all (97%) of the phosphosites that were responsive to exercise are annotated in the PhosphoSitePlus database but, importantly, the majority of these have not previously been associated with the cardiac response to exercise. More than two-thirds of the exercise-responsive phosphosites were different from those identified in previous phosphoproteome profiling of the cardiac response to β1-adrenergic receptor stimulation. Moreover, we report entirely new phosphorylation sites on 4 cardiac proteins, including S81 of muscle LIM protein, and identified 7 exercise-responsive kinases, including myofibrillar protein kinases such as obscurin, titin and the striated-muscle-specific serine/threonine kinase (SPEG) that may be worthwhile targets for future investigation

On the Rate of Synthesis of Individual Proteins within and between Different Striated Muscles of the Rat

The turnover of muscle protein is responsive to different (patho)-physiological conditions but little is known about the rate of synthesis at the level of individual proteins or whether this varies between different muscles. We investigated the synthesis rate of eight proteins (actin, albumin, ATP synthase alpha, beta enolase, creatine kinase, myosin essential light chain, myosin regulatory light chain and tropomyosin) in the extensor digitorum longus, diaphragm, heart and soleus of male Wistar rats (352 ± 30 g body weight). Animals were assigned to four groups (n = 3, in each), including a control and groups that received deuterium oxide (2H2O) for 4 days, 7 days or 14 days. Deuterium labelling was initiated by an intraperitoneal injection of 10 μL/g body weight of 99.9% 2H2O-saline, and was maintained by administration of 5% (v/v) 2H2O in drinking water provided ad libitum. Homogenates of the isolated muscles were analysed by 2-dimensional gel electrophoresis and matrix-assisted laser desorption ionisation time of flight mass spectrometry. Proteins were identified against the SwissProt database using peptide mass fingerprinting. For each of the eight proteins investigated, the molar percent enrichment (MPE) of 2H and rate constant (k) of protein synthesis was calculated from the mass isotopomer distribution of peptides based on the amino acid sequence and predicted number of exchangeable C–H bonds. The average MPE (2.14% ± 0.2%) was as expected and was consistent across muscles harvested at different times (i.e., steady state enrichment was achieved). The synthesis rate of individual proteins differed markedly within each muscle and the rank-order of synthesis rates differed among the muscles studied. After 14 days the fraction of albumin synthesised (23% ± 5%) was significantly (p < 0.05) greater than for other muscle proteins. These data represent the first attempt to study the synthesis rates of individual proteins across a number of different striated muscles

Degradation of ribosomal and chaperone proteins is attenuated during the differentiation of replicatively aged C2C12 myoblasts

Background: Cell assays are important for investigating the mechanisms of ageing, including losses in protein homeostasis and ‘proteostasis collapse’. We used novel isotopic labelling and proteomic methods to investigate protein turnover in replicatively aged (>140 population doublings) murine C2C12 myoblasts that exhibit impaired differentiation and serve as a model for age-related declines in muscle homeostasis. Methods: The Absolute Dynamic Profiling Technique for Proteo-mics (Proteo-ADPT) was used to investigate proteostasis in young (passage 6-10) and replicatively aged (passage 48-50) C2C12 myoblast cultures supplemented with deuterium oxide (D2O) during early (0 h – 24 h) or late (72 h – 96 h) periods of differentiation. Peptide mass spectrometry was used to quantify the absolute rates of abundance change, synthesis and degradation of individual proteins. Results: Young cells exhibited a consistent ~25% rise in protein accretion over the 96-h experimental period. In aged cells, protein accretion increased by 32% (P<0.05) during early differentiation, but then fell back to baseline levels by 96-h. Proteo-ADPT encompassed 116 proteins and 74 proteins exhibited significantly (P<0.05, FDR <5% interaction between age * differentiation stage) different changes in abundance between young and aged cells at early and later periods of differentiation, including proteins associated with translation, glycolysis, cell-cell adhesion, ribosomal biogenesis and the regulation of cell shape. During early differentiation, heat shock and ribosomal protein abundances increased in aged cells due to suppressed degradation rather than heightened synthesis. For instance, HS90A increased at a rate of 10.62 ± 1.60 ng/well/h in aged which was significantly greater than the rate of accretion (1.86 ± 0.49 ng/well/h) in young cells. HS90A synthesis was similar in young (21.23 ± 3.40 ng/well/h) and aged (23.69 ± 1.13 ng/well/h) but HS90A degradation was significantly (P = 0.05) greater in young (19.37 ± 2.93 ng/well/h) versus aged (13.06 ± 0.76 ng/well/h) cells. During later differentiation the HS90A degradation (8.94 ± 0.38 ng/well/h) and synthesis (7.89 ± 1.28 ng/well/h) declined and were significantly less than the positive net balance between synthesis and degradation (ASR = 28.14 ± 3.70 ng/well/h vs ADR = 21.49 ± 3.13 ng/well/h) in young cells. Conclusion: Our results suggest a loss of proteome quality as a precursor to the lack of fusion of aged myoblasts. The quality of key chaperone proteins, including HS90A, HS90B and HSP7C was reduced in aged cells and may account for the disruption to cell signalling required for the later stages of differentiation and fusion

Label-free profiling of white adipose tissue of rats exhibiting high or low levels of intrinsic exercise capacity

Divergent selection has created rat phenotypes of high- and low-capacity runners (HCR and LCR, respectively) that have differences in aerobic capacity and correlated traits such as adiposity. We analyzed visceral adipose tissue of HCR and LCR using label-free high-definition MS (elevated energy) profiling. The running capacity of HCR was ninefold greater than LCR. Proteome profiling encompassed 448 proteins and detected 30 significant (p <0.05; false discovery rate <10%, calculated using q-values) differences. Approximately half of the proteins analyzed were of mitochondrial origin, but there were no significant differences in the abundance of proteins involved in aerobic metabolism. Instead, adipose tissue of LCR rats exhibited greater abundances of proteins associated with adipogenesis (e.g. cathepsin D), ER stress (e.g. 78 kDa glucose response protein), and inflammation (e.g. Ig gamma-2B chain C region). Whereas the abundance antioxidant enzymes such as superoxide dismutase [Cu-Zn] was greater in HCR tissue. Putative adipokines were also detected, in particular protein S100-B, was 431% more abundant in LCR adipose tissue. These findings reveal low running capacity is associated with a pathological profile in visceral adipose tissue proteome despite no detectable differences in mitochondrial protein abundance

The application of proteomics in muscle exercise physiology

Introduction: Exercise offers protection from non-communicable diseases and extends healthspan by offsetting natural physiological declines that occur in older age. Striated muscle is the largest bodily organ; it underpins the capacity for physical work, and the responses of muscle to exercise convey the health benefits of a physically active lifestyle. Proteomic surveys of muscle provide a means to study the protective effects of exercise and this review summaries some key findings from literature listed in PubMed during the last 10 years that have led to new insight in muscle exercise physiology. Areas covered: ‘Bottom-up’ analyses involving liquid-chromatography tandem mass spectrometry (LCMS/MS) of peptide digests have become the mainstay of proteomic studies and have been applied to muscle mitochondrial fractions. Enrichment techniques for post-translational modifications, including phosphorylation, acetylation and ubiquitination, have evolved and the analysis of site-specific modifications has become a major area of interest in exercise proteomics. Finally, we consider emergent techniques for dynamic analysis of muscle proteomes that offer new insight to protein turnover and the contributions of synthesis and degradation to changes in protein abundance in response to exercise training. Expert opinion: Burgeoning methods for dynamic proteome profiling offer new opportunities to study the mechanisms of muscle adaptation

Fractional Synthesis Rates of Individual Proteins in Rat Soleus and Plantaris Muscles.

Differences in the protein composition of fast- and slow-twitch muscle may be maintained by different rates of protein turnover. We investigated protein turnover rates in slow-twitch soleus and fast-twitch plantaris of male Wistar rats (body weight 412 ± 69 g). Animals were assigned to four groups (n = 3, in each), including a control group (0 d) and three groups that received deuterium oxide (D2O) for either 10 days, 20 days or 30 days. D2O administration was initiated by an intraperitoneal injection of 20 μL of 99% D2O-saline per g body weight, and maintained by provision of 4% (v/v) D2O in the drinking water available ad libitum. Soluble proteins from harvested muscles were analysed by liquid chromatography-tandem mass spectrometry and identified against the SwissProt database. The enrichment of D2O and rate constant (k) of protein synthesis was calculated from the abundance of peptide mass isotopomers. The fractional synthesis rate (FSR) of 44 proteins in soleus and 34 proteins in plantaris spanned from 0.58%/day (CO1A1: Collagen alpha-1 chain) to 5.40%/day NDRG2 (N-myc downstream-regulated gene 2 protein). Eight out of 18 proteins identified in both muscles had a different FSR in soleus than in plantaris (p < 0.05)

Dynamic Profiling of Protein Mole Synthesis Rates During C2C12 Myoblast Differentiation

Mole (MSR) and fractional (FSR) synthesis rates of proteins during C2C12 myoblast differentiation were investigated. Myoblast cultures supplemented with D2O during 0–24 h or 72–96 h of differentiation were analysed by LC‐MS/MS to calculate protein FSR and MSR after samples were spiked with yeast alcohol dehydrogenase (ADH1). Profiling of 153 proteins detected 70 significant (p ≤ 0.05, FDR ≤1 %) differences in abundance between cell states. Early differentiation was enriched by clusters of ribosomal and heat shock proteins, whereas later differentiation was associated with actin filament binding. The median (first ‐ third quartile) FSR (%/h) during early differentiation 4.1 (2.7‐5.3) was ∼2‐fold greater than later differentiation 1.7 (1.0‐2.2), equating to MSR of 0.64, (0.38‐1.2) and 0.28, (0.1‐0.5) fmol/h/ug total protein, respectively. MSR corresponded more closely with abundance data and highlighted proteins associated with glycolytic processes and intermediate filament protein binding that were not evident amongst FSR data. Similarly, MSR during early differentiation accounted for 78 % of the variation in protein abundance during later differentiation, whereas FSR accounted for 4%. Conclusively, the interpretation of protein synthesis data differed when reported in mole or fractional terms, which has consequences when studying the allocation of cellular resources

Time-of-day variation on performance measures in repeated-sprint tests: A systematic review.

The lack of standardization of methods and procedures have hindered agreement in the literature related to time-of-day effects on repeated sprint performance and needs clarification. Therefore, the aim of the present study was to investigate and systematically review the evidence relating to time-of-day based on performance measures in repeated-sprints. The entire content of PubMed (MEDLINE), Scopus, SPORTDiscus® (via EBSCOhost) and Web of Science was searched. Only experimental research studies conducted in male adult participants aged ≥18yrs, published in English before June 2019 were included. Studies assessing repeated-sprints between a minimum of two time-points during the day (morning versus evening) were deemed eligible. The primary search revealed that a total of 10 out of 112 articles were considered eligible and subsequently included. Seven articles were deemed strong and three moderate quality. Eight studies found repeated-sprint performance across the first, first few, or all sprints, to increase in favor of the evening. The magnitude of difference is dependent on the modality and the exercise protocol used. The non-motorized treadmill established an average 3.5–8.5% difference in distance covered, average and peak velocity, and average power, across all sprints in three studies and in peak power in two studies. In cycling, power output differed across all sprints by 6.0% in one study and 8.0% for the first sprint only in five studies. All four studies measuring power decrement values (i.e. rate of fatigue) established differences up to 4.0% and two out of five studies established total work to be significantly higher by 8.0%. Repeated-sprint performance is affected by time-of-day with greater performance in the late/early afternoon. The magnitude is dependent on the variable assessed and the mode of exercise. There is a clear demand for more rigorous investigations which control factors that specifically relate to investigations of time-of-day and are specific to the sport of individuals

Endurance-Type Exercise Increases Bulk and Individual Mitochondrial Protein Synthesis Rates in Rats.

Physical activity increases muscle protein synthesis rates. However, the impact of exercise on the coordinated up- and/or downregulation of individual protein synthesis rates in skeletal muscle tissue remains unclear. The authors assessed the impact of exercise on mixed muscle, myofibrillar, and mitochondrial protein synthesis rates as well as individual protein synthesis rates in vivo in rats. Adult Lewis rats either remained sedentary (n = 3) or had access to a running wheel (n = 3) for the last 2 weeks of a 3-week experimental period. Deuterated water was injected and subsequently administered in drinking water over the experimental period. Blood and soleus muscle were collected and used to assess bulk mixed muscle, myofibrillar, and mitochondrial protein synthesis rates using gas chromatography-mass spectrometry and individual muscle protein synthesis rates using liquid chromatography-mass spectrometry (i.e., dynamic proteomic profiling). Wheel running resulted in greater myofibrillar (3.94 ± 0.26 vs. 3.03 ± 0.15%/day; p < .01) and mitochondrial (4.64 ± 0.24 vs. 3.97 ± 0.26%/day; p < .05), but not mixed muscle (2.64 ± 0.96 vs. 2.38 ± 0.62%/day; p = .71) protein synthesis rates, when compared with the sedentary condition. Exercise impacted the synthesis rates of 80 proteins, with the difference from the sedentary condition ranging between -64% and +420%. Significantly greater synthesis rates were detected for F1-ATP synthase, ATP synthase subunit alpha, hemoglobin, myosin light chain-6, and synaptopodin-2 (p < .05). The skeletal muscle protein adaptive response to endurance-type exercise involves upregulation of mitochondrial protein synthesis rates, but it is highly coordinated as reflected by the up- and downregulation of various individual proteins across different bulk subcellular protein fractions