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.

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.

Montmorency tart cherry juice does not reduce markers of muscle soreness, function and inflammation following professional male rugby League match-play.

Rugby League (RL) match-play causes muscle damage, inflammation and symptoms of fatigue. To facilitate recovery, nutritional interventions are often employed, including Montmorency cherry juice (MC). We assessed the effects of MC on recovery following RL match-play in eleven male professional RL players who played in two matches (7-days apart) with MC or placebo (PLB) supplemented for 5-days pre-match, matchday and 2-days post-match. Blood was collected 48h pre-match, half-time, within 30-mins of full-time and 48h post-match to assess Interleukin concentrations (IL-6, -8 -10). Self-reported sleep, fatigue, mood, stress, and muscle-soreness were assessed 24h pre and 24 and 48h post-matches with muscle function assessed 48h pre and 48h post-match. No differences in distance covered (6334 ± 1944 Vs 6596 ± 1776m) and total collisions (28 ± 11 Vs 29 ± 13) were observed between both matches. There was a small albeit significant increase in IL-6, -8 and -10 concentrations pre to post-match in both PLB (IL-6: 0.83 ± 0.92 Vs 2.91 ± 1.40, IL-8: 2.16 ± 1.22 Vs 3.91 ± 1.61 and IL-10: 2.51 ± 2.14 Vs 0.61 ± 0.50 pg.mL-1) and MC groups (IL-6: 0.53 ± 0.53 Vs 2.24 ± 1.73, IL-8: 1.85 ± 0.96 Vs 3.46 ± 1.12 and IL-10: 0.48 ± 0.50 Vs 2.54 ± 2.10 pg.mL-1), although there were no significant differences between groups (P<0.05). Likewise, there was a small but significant increase in muscle soreness (P=0.01) and reduction in CMJ (P=0.003) with no significant differences between groups. No significant changes in sleep, fatigue or mood (P>0.05) were observed pre to post-match or between groups. These data suggest MC does not affect the modest changes observed in cytokine responses and markers of recovery from RL match-play.Keywords: Team Sport, Nutrition, Performance, Recovery.

Exercising Bioengineered Skeletal Muscle In Vitro: Biopsy to Bioreactor.

The bioengineering of skeletal muscle tissue in-vitro has enabled researchers to more closely mimic the in-vivo skeletal muscle niche. The three-dimensional (3-D) structure of the tissue engineered systems employed to date enable the generation of highly aligned and differentiated myofibers within a representative biological matrix. The use of electrical stimulation to model concentric contraction, via innervation of the myofibers, and the use of mechanical loading to model passive lengthening or stretch has begun to provide a manipulable environment to investigate the cellular and molecular responses following exercise mimicking stimuli in-vitro. Currently available bioreactor systems allow either electrical stimulation or mechanical loading to be utilized at any given time. In the present manuscript, we describe in detail the methodological procedures to create 3-D bioengineered skeletal muscle using both cell lines and/or primary human muscle derived cells from a tissue biopsy, through to modeling exercising stimuli using a bioreactor that can provide both electrical stimulation and mechanical loading simultaneously within the same in-vitro system.

Murine myoblast migration: influence of replicative ageing and nutrition.

Cell migration is central to skeletal muscle repair following damage. Leucine and ß-Hydroxy ß-methylbutyric acid (HMB) are supplements consumed for recovery from muscle damaging exercise in humans, however, their impact on muscle cell migration with age is not yet understood. We hypothesised that replicatively aged ("aged"; P46-P48) myoblasts would be less efficient at basal and supplemented repair versus parental controls ("control"; P12-P16). Aged and control myoblasts were scratch-damaged and migration velocity, directionality and distance assessed over 48 h in the absence and presence of leucine (10 mM) or HMB (10 mM) ± PI3K/Akt (LY294002 10 µM), ERK (PD98059 5 µM) or mTOR (rapamycin 0.5 µM) inhibition. Opposing our hypothesis, aged cells displayed increased velocities, directionality and distance migrated (P < 0.001) versus control. Leucine and HMB significantly increased (P < 0.001) the same parameters in control cells. The supplements were with smaller, albeit significant impact on aged cell velocity (P < 0.001) and in the presence of HMB only, distance (P = 0.041). Inhibitor studies revealed that, PI3K and ERK activation were essential for velocity, directionality and migration distance of aged cells in basal conditions, whereas mTOR was important for directionality only. While PI3K activation was critical for all parameters in control cells (P < 0.001), inhibition of ERK or mTOR improved, rather than reduced, control cell migration distance. Enhanced basal velocity, directionality and distance in aged cells required ERK and PI3K activation. By contrast, in control cells, basal migration was underpinned by PI3K activation, and facilitated by leucine or HMB supplementation, to migration levels seen in aged cells. These data suggest that replicatively aged myoblasts are not anabolically resistant per se, but are capable of efficient repair, underpinned by altered signaling pathways, compared with unaged control myoblasts.