Facioscapulohumeral Muscular Dystrophy is Associated With Altered Myoblast Proteome Dynamics.

Proteomic studies in facioscapulohumeral muscular dystrophy (FSHD) could offer new insight into disease mechanisms underpinned by post-transcriptional processes. We used stable isotope (deuterium oxide; D2O) labeling and peptide mass spectrometry to investigate the abundance and turnover rates of proteins in cultured muscle cells from two individuals affected by FSHD and their unaffected siblings (UASb). We measured the abundance of 4420 proteins and the turnover rate of 2324 proteins in each (n = 4) myoblast sample. FSHD myoblasts exhibited a greater abundance but slower turnover rate of subunits of mitochondrial respiratory complexes and mitochondrial ribosomal proteins, which may indicate an accumulation of "older" less viable mitochondrial proteins in myoblasts from individuals affected by FSHD. Treatment with a 2'-O-methoxyethyl modified antisense oligonucleotide targeting exon 3 of the double homeobox 4 (DUX4) transcript tended to reverse mitochondrial protein dysregulation in FSHD myoblasts, indicating the effect on mitochondrial proteins may be a DUX4-dependent mechanism. Our results highlight the importance of post-transcriptional processes and protein turnover in FSHD pathology and provide a resource for the FSHD research community to explore this burgeoning aspect of FSHD.

People with obesity exhibit losses in muscle proteostasis that are partly improved by exercise training.

This pilot experiment examines if a loss in muscle proteostasis occurs in people with obesity and whether endurance exercise positively influences either the abundance profile or turnover rate of proteins in this population. Men with (n = 3) or without (n = 4) obesity were recruited and underwent a 14-d measurement protocol of daily deuterium oxide (D2 O) consumption and serial biopsies of vastus lateralis muscle. Men with obesity then completed 10-weeks of high-intensity interval training (HIIT), encompassing 3 sessions per week of cycle ergometer exercise with 1 min intervals at 100% maximum aerobic power interspersed by 1 min recovery periods. The number of intervals per session progressed from 4 to 8, and during weeks 8-10 the 14-d measurement protocol was repeated. Proteomic analysis detected 352 differences (p < 0.05, false discovery rate < 5%) in protein abundance and 19 (p < 0.05) differences in protein turnover, including components of the ubiquitin-proteasome system. HIIT altered the abundance of 53 proteins and increased the turnover rate of 22 proteins (p < 0.05) and tended to benefit proteostasis by increasing muscle protein turnover rates. Obesity and insulin resistance are associated with compromised muscle proteostasis, which may be partially restored by endurance exercise.

Dynamic Profiling of Protein Mole Synthesis Rates during C2C12 Myoblast Differentiation.

Mole (MSR) and fractional (FSR) synthesis rates of proteins during C2C12 myoblast differentiation are investigated. Myoblast cultures supplemented with D2 O during 0-24 h or 72-96 h of differentiation are analyzed by LC-MS/MS to calculate protein FSR and MSR after samples are 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 is enriched by clusters of ribosomal and heat shock proteins, whereas later differentiation is associated with actin filament binding. The median (first-third quartile) FSR (%/h) during early differentiation 4.1 (2.7-5.3) is approximately twofold 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-1  µg-1 total protein, respectively. MSR corresponds more closely with abundance data and highlights proteins associated with glycolytic processes and intermediate filament protein binding that are not evident among FSR data. Similarly, MSR during early differentiation accounts for 78% of the variation in protein abundance during later differentiation, whereas FSR accounts for 4%. Conclusively, the interpretation of protein synthesis data differs when reported in mole or fractional terms, which has consequences when studying the allocation of cellular resources.

Adaptation of rat fast-twitch muscle to endurance activity is underpinned by changes to protein degradation as well as protein synthesis.

Muscle adaptations to exercise are underpinned by alterations to the abundance of individual proteins, which may occur through a change either to the synthesis or degradation of each protein. We used deuterium oxide (2 H2 O) labeling and chronic low-frequency stimulation (CLFS) in vivo to investigate the synthesis, abundance, and degradation of individual proteins during exercise-induced muscle adaptation. Independent groups of rats received CLFS (10 Hz, 24 h/d) and 2 H2 O for 0, 10, 20, or 30 days. The extensor digitorum longus (EDL) was isolated from stimulated (Stim) and contralateral non-stimulated (Ctrl) legs. Proteomic analysis encompassed 38 myofibrillar and 46 soluble proteins and the rates of change in abundance, synthesis, and degradation were reported in absolute (ng/d) units. Overall, synthesis and degradation made equal contributions to the adaptation of the proteome, including instances where a decrease in protein-specific degradation primarily accounted for the increase in abundance of the protein.

Reliability of Protein Abundance and Synthesis Measurements in Human Skeletal Muscle.

The repeatability of dynamic proteome profiling (DPP), which is a novel technique for measuring the relative abundance (ABD) and fractional synthesis rate (FSR) of proteins in humans, is investigated. LC-MS analysis is performed on muscle samples taken from male participants (n = 4) that consumed 4 × 50 mL doses of deuterium oxide (2 H2 O) per day for 14 days. ABD is measured by label-free quantitation and FSR is calculated from time-dependent changes in peptide mass isotopomer abundances. One-hundred one proteins have at least one unique peptide and are used in the assessment of protein ABD. Fifty-four of these proteins meet more stringent criteria and are used in the assessment of FSR data. The median (M), lower-, (Q1 ) and upper-quartile (Q3 ) values for protein FSR (%/d) are M = 1.63, Q1  = 1.07, and Q3  = 3.24, respectively. The technical CV of ABD data has a median value of 3.6% (Q1 1.7% to Q3 6.7%), whereas the median CV of FSR data is 10.1% (Q1 3.5% to Q3 16.5%). These values compare favorably against other assessments of technical repeatability of proteomics data, which often set a CV of 20% as the upper bound of acceptability.

Striated muscle-specific serine/threonine-protein kinase beta segregates with high versus low responsiveness to endurance exercise training.

Bidirectional selection for either high or low responsiveness to endurance running has created divergent rat phenotypes of high-response trainers (HRT) and low-response trainers (LRT). We conducted proteome profiling of HRT and LRT gastrocnemius of 10 female rats (body weight 279 ± 35 g; n = 5 LRT and n = 5 HRT) from generation 8 of selection. Differential analysis of soluble proteins from gastrocnemius was conducted by label-free quantitation. Genetic association studies were conducted in 384 Russian international-level athletes (age 23.8 ± 3.4 yr; 202 men and 182 women) stratified to endurance or power disciplines. Proteomic analysis encompassed 1,024 proteins, 76 of which exhibited statistically significant (P < 0.05, false discovery rate <1%) differences between HRT and LRT muscle. There was significant enrichment of enzymes involved in glycolysis/gluconeogenesis in LRT muscle but no enrichment of gene ontology phrases in HRT muscle. Striated muscle-specific serine/threonine-protein kinase-beta (SPEG-ß) exhibited the greatest difference in abundance and was 2.64-fold greater (P = 0.0014) in HRT muscle. Coimmunoprecipitation identified 24 potential binding partners of SPEG-ß in HRT muscle. The frequency of the G variant of the rs7564856 polymorphism that increases SPEG gene expression was significantly greater (32.9 vs. 23.8%; OR = 1.6, P = 0.009) in international-level endurance athletes (n = 258) compared with power athletes (n = 126) and was significantly associated (ß = 8.345, P = 0.0048) with a greater proportion of slow-twitch fibers in vastus lateralis of female endurance athletes. Coimmunoprecipitation of SPEG-ß in HRT muscle discovered putative interacting proteins that link with previously reported differences in transforming growth factor-ß signaling in exercised muscle.

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 (LC-MS/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.

Sleep and the athlete: narrative review and 2021 expert consensus recommendations.

Elite athletes are particularly susceptible to sleep inadequacies, characterised by habitual short sleep (<7 hours/night) and poor sleep quality (eg, sleep fragmentation). Athletic performance is reduced by a night or more without sleep, but the influence on performance of partial sleep restriction over 1-3 nights, a more real-world scenario, remains unclear. Studies investigating sleep in athletes often suffer from inadequate experimental control, a lack of females and questions concerning the validity of the chosen sleep assessment tools. Research only scratches the surface on how sleep influences athlete health. Studies in the wider population show that habitually sleeping <7 hours/night increases susceptibility to respiratory infection. Fortunately, much is known about the salient risk factors for sleep inadequacy in athletes, enabling targeted interventions. For example, athlete sleep is influenced by sport-specific factors (relating to training, travel and competition) and non-sport factors (eg, female gender, stress and anxiety). This expert consensus culminates with a sleep toolbox for practitioners (eg, covering sleep education and screening) to mitigate these risk factors and optimise athlete sleep. A one-size-fits-all approach to athlete sleep recommendations (eg, 7-9 hours/night) is unlikely ideal for health and performance. We recommend an individualised approach that should consider the athlete's perceived sleep needs. Research is needed into the benefits of napping and sleep extension (eg, banking sleep).

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.

Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.

It is generally accepted that muscle adaptation to resistance exercise (REX) training is underpinned by contraction-induced, increased rates of protein synthesis and dietary protein availability. By using dynamic proteome profiling (DPP), we investigated the contribution of both synthesis and breakdown to changes in abundance on a protein-by-protein basis in human skeletal muscle. Age-matched, overweight males consumed 9 d of a high-fat, low-carbohydrate diet during which time they either undertook 3 sessions of REX or performed no exercise. Precursor enrichment and the rate of incorporation of deuterium oxide into newly synthesized muscle proteins were determined by mass spectrometry. Ninety proteins were included in the DPP, with 28 proteins exhibiting significant responses to REX. The most common pattern of response was an increase in turnover, followed by an increase in abundance with no detectable increase in protein synthesis. Here, we provide novel evidence that demonstrates that the contribution of synthesis and breakdown to changes in protein abundance induced by REX differ on a protein-by-protein basis. We also highlight the importance of the degradation of individual muscle proteins after exercise in human skeletal muscle.-Camera, D. M., Burniston, J. G., Pogson, M. A., Smiles, W. J., Hawley, J. A. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.

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-1min-1 and 69.8±5mlkg-1min-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 ß1-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.

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.

Exercise improves mitochondrial and redox-regulated stress responses in the elderly: better late than never!

Ageing is associated with several physiological declines to both the cardiovascular (e.g. reduced aerobic capacity) and musculoskeletal system (muscle function and mass). Ageing may also impair the adaptive response of skeletal muscle mitochondria and redox-regulated stress responses to an acute exercise bout, at least in mice and rodents. This is a functionally important phenomenon, since (1) aberrant mitochondrial and redox homeostasis are implicated in the pathophysiology of musculoskeletal ageing and (2) the response to repeated exercise bouts promotes exercise adaptations and some of these adaptations (e.g. improved aerobic capacity and exercise-induced mitochondrial remodelling) offset age-related physiological decline. Exercise-induced mitochondrial remodelling is mediated by upstream signalling events that converge on downstream transcriptional co-factors and factors that orchestrate a co-ordinated nuclear and mitochondrial transcriptional response associated with mitochondrial remodelling. Recent translational human investigations have demonstrated similar exercise-induced mitochondrial signalling responses in older compared with younger skeletal muscle, regardless of training status. This is consistent with data indicating normative mitochondrial remodelling responses to long-term exercise training in the elderly. Thus, human ageing is not accompanied by diminished mitochondrial plasticity to acute and chronic exercise stimuli, at least for the signalling pathways measured to date. Exercise-induced increases in reactive oxygen and nitrogen species promote an acute redox-regulated stress response that manifests as increased heat shock protein and antioxidant enzyme content. In accordance with previous reports in rodents and mice, it appears that sedentary ageing is associated with a severely attenuated exercise-induced redox stress response that might be related to an absent redox signal. In this regard, regular exercise training affords some protection but does not completely override age-related defects. Despite some failed redox-regulated stress responses, it seems mitochondrial responses to exercise training are intact in skeletal muscle with age and this might underpin the protective effect of exercise training on age-related musculoskeletal decline. Whilst further investigation is required, recent data suggest that it is never too late to begin exercise training and that lifelong training provides protection against several age-related declines at both the molecular (e.g. reduced mitochondrial function) and whole-body level (e.g. aerobic capacity).

Label-free profiling of skeletal muscle using high-definition mass spectrometry.

We report automated and time-efficient (2 h per sample) profiling of muscle using ultra-performance LC coupled directly with high-definition MS (HDMS(E)). Soluble proteins extracted from rat gastrocnemius (n = 10) were digested with trypsin and analyzed in duplicate using a 90 min RPLC gradient. Protein identification and label-free quantitation were performed from HDMS(E) spectra analyzed using Progenesis QI for Proteomics software. In total 1514 proteins were identified. Of these, 811 had at least three unique peptides and were subsequently used to assess the dynamic range and precision of LC-HDMS(E) label-free profiling. Proteins analyzed by LC-HDMS(E) encompass the entire complement of glycolytic, ß-oxidation, and tricarboxylic acid enzymes. In addition, numerous components of the electron transport chain and protein kinases involved in skeletal muscle regulation were detected. The dynamic range of protein abundances spanned four orders of magnitude. The correlation between technical replicates of the ten biological samples was R(2) = 0.9961 ± 0.0036 (95% CI = 0.9940 - 0.9992) and the technical CV averaged 7.3 ± 6.7% (95% CI = 6.87 - 7.79%). This represents the most sophisticated label-free profiling of skeletal muscle to date.

Urinary proteomics in obstructive sleep apnoea and obesity.

BACKGROUND: Obstructive sleep apnoea (OSA) is a common complication of obesity and can have a substantial negative impact on a patient's quality of life and risk of cardiovascular disease. The aim of this case-control study was to undertake discovery profiling of urinary peptides using capillary electrophoresis-mass spectrometry (CE-MS) in obese subjects with and without OSA, without a history of coronary artery disease. MATERIALS AND METHODS: Urinary samples were analysed by CE-MS. Body composition and blood pressure measurements were recorded. Overnight polysomnography was conducted to confirm or refute OSA. OSA patients were naïve to continuous positive airway pressure treatment. RESULTS: Sixty-one subjects with OSA (age 47 ± 9 years, BMI 43 ± 8 kg/m(2)) and 31 controls (age 49 ± 10 years, BMI 40 ± 5 kg/m(2)) were studied; P = ns for age and BMI. Apnoea-hypopnoea Index was higher in patients with OSA (24 ± 18·6) than controls without OSA (non-OSA) (2·6 ± 1·1; P < 0·0001). Metabolic syndrome was present in 35 (57%) of those with OSA compared with 4 (13%) of controls (P < 0·0001). Twenty-four polypeptides were candidates for differential distribution (P < 0·01), although these differences did not reach significance after multiple testing. Sequences were determined for eight peptides demonstrating origins from collagens and fibrinogen alpha. CONCLUSIONS: In this study, we report for the first time, urinary proteomic profile analyses using CE-MS in OSA and non-OSA obese groups. The differences in urinary proteomic profiles prior to adjustment for multiple testing, with increased metabolic syndrome in obese OSA subjects, suggest that there may be a role for CE-MS in characterising urinary profiles in severely obese populations with OSA.

Circadian variation in muscle force output in males using isokinetic, isometric dynamometry: can we observe this in multi-joint movements using the muscleLab force-velocity encoder and are they similar in peak and magnitude?

We have investigated the magnitude of circadian variation in Isokinetic and Isometric strength of the knee extensors and flexors, as well as back squat and bench press performance using the MuscleLab force velocity transducer. Ten resistance-trained males (mean±SD: age 21.5 ± 1.1 years; body mass 78.3 ± 5.2 kg; height 1.71 ± 0.07 m) underwent a) three to four familiarization sessions on each dynamometer and b) four sessions at different times of day (03:00, 09:00, 15:00 and 21:00 h). Each session was administered in a counterbalanced order and included a period when Perceived onset of mood states (POMS), then rectal and muscle temperature (Trec, Tm) was measured at rest, after which a 5-min standardized 150 W warm-up was performed on a cycle ergometer. Once completed, Isokinetic (60 and 240°·s-1 for extension and flexion) and Isometric dynamometry with peak torque (PT), time-to-peak-torque (tPT) and peak force (PF) and % activation was measured. Lastly, Trec and Tm were measured before the bench press (at 30, 50 and 70 kg) and back squat (at 40, 60 and 80 kg) exercises. A linear encoder was attached to an Olympic bar used for the exercises and average force (AF), peak velocity (PV) and time-to-peak-velocity (tPV) were measured (MuscleLab software; MuscleLab Technology, Langesund, Norway) during the concentric phase of the movements. Five-min recovery was allowed between each set with three repetitions being completed. General linear models with repeated measures and cosinor analysis were used to analyse the data. Values for Trec and Tm at rest were higher in the evening compared to morning values (Acrophase Φ: 16:35 and 17:03 h, Amplitude A: 0.30 and 0.23°C, Mesor M: 36.64 and 37.43°C, p < 0.05). Vigor, happy and fatigue mood states responses showed Φ 16:11 and 16:03 h and 02:05 h respectively. Circadian rhythms were apparent for all variables irrespective of equipment used where AF, PF and PT values peaked between 16:18 and 18:34 h; PV, tPV and tPT peaked between 05:54 and 08:03 h (p < 0.05). In summary, circadian rhythms in force output (force, torque, power, and velocity) were shown for isokinetic, isometric dynamometers and complex multi-joint movements (using a linear encoder); where tPV and tPT occur in the morning compared to the evening. Circadian rhythms in strength can be detected using a portable, low-cost instrument that shows similar cosinor characteristics as established dynamometers. Hence, muscle-strength can be measured in a manner that is more directly transferable to the world of athletic and sports performance.

Gene expression profiling of gastrocnemius of "minimuscle" mice.

Few studies have investigated heterogeneity of selection response in replicate lines subjected to equivalent selection. We developed four replicate lines of mice based on high levels of voluntary wheel running (high runner or HR lines) while also maintaining four nonselected control lines. This led to the unexpected discovery of the HR minimuscle (HRmini) phenotype, recognized by a 50% reduction in hindlimb muscle mass, which became fixed in 1 of the four HR selected lines. Here, we report genome-wide expression profiling describing transcriptome differences between HRnormal and HRmini medial gastrocnemius. Consistent with the known reduction of type IIB fibers in HRmini, Myh4 gene expression was -8.82-fold less (P = 0.0001) in HRmini, which was closely associated with differences in the "calcium signaling" canonical pathway, including structural genes (e.g., Mef2c, twofold greater in HRmini, P = 0.0003) and myogenic factors (e.g., Myog, 3.8-fold greater in HRmini, P = 0.0026) associated with slow-type myofibers. The gene that determines the HRmini phenotype is known to reside in a 2.6335-Mb interval on mouse chromosome 11 and 7 genes (Myh10, Chrnb1, Acadvl, Senp3, Gabarap, Eif5a, and Clec10a) from this region were differentially expressed. Verification by real-time PCR confirmed 1.5-fold greater (P < 0.05) expression of very long chain acyl-CoA dehydrogenase (Acadvl) in HRmini. Ten other genes associated with fatty acid metabolism were also upregulated in HRmini, suggesting differences in the ability to metabolize fatty acids in HRnormal and HRmini muscles. This work provides a resource for understanding differences in muscle phenotypes in populations exhibiting high running capacity.

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 Proteomics (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 (D2 O) during early (0-24 h) or late (72-96 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 (synthesis = 28.14 ± 3.70 ng/well/h vs. degradation = 21.49 ± 3.13 ng/well/h) in young cells. CONCLUSIONS: 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.

Genome-Wide Association Study Identifies CDKN1A as a Novel Locus Associated with Muscle Fiber Composition.

Muscle fiber composition is associated with physical performance, with endurance athletes having a high proportion of slow-twitch muscle fibers compared to power athletes. Approximately 45% of muscle fiber composition is heritable, however, single nucleotide polymorphisms (SNP) underlying inter-individual differences in muscle fiber types remain largely unknown. Based on three whole genome SNP datasets, we have shown that the rs236448 A allele located near the cyclin-dependent kinase inhibitor 1A (CDKN1A) gene was associated with an increased proportion of slow-twitch muscle fibers in Russian (n = 151; p = 0.039), Finnish (n = 287; p = 0.03), and Japanese (n = 207; p = 0.008) cohorts (meta-analysis: p = 7.9 × 10−5. Furthermore, the frequency of the rs236448 A allele was significantly higher in Russian (p = 0.045) and Japanese (p = 0.038) elite endurance athletes compared to ethnically matched power athletes. On the contrary, the C allele was associated with a greater proportion of fast-twitch muscle fibers and a predisposition to power sports. CDKN1A participates in cell cycle regulation and is suppressed by the miR-208b, which has a prominent role in the activation of the slow myofiber gene program. Bioinformatic analysis revealed that the rs236448 C allele was associated with increased CDKN1A expression in whole blood (p = 8.5 × 10−15) and with greater appendicular lean mass (p = 1.2 × 10−5), whereas the A allele was associated with longer durations of exercise (p = 0.044) reported amongst the UK Biobank cohort. Furthermore, the expression of CDKN1A increased in response to strength (p < 0.0001) or sprint (p = 0.00035) training. Accordingly, we found that CDKN1A expression is significantly (p = 0.002) higher in the m. vastus lateralis of strength athletes compared to endurance athletes and is positively correlated with the percentage of fast-twitch muscle fibers (p = 0.018). In conclusion, our data suggest that the CDKN1A rs236448 SNP may be implicated in the determination of muscle fiber composition and may affect athletic performance.