Age- and activity-related differences in the abundance of Myosin essential and regulatory light chains in human muscle

Traditional methods for phenotyping skeletal muscle (e.g., immunohistochemistry) are labor-intensive and ill-suited to multixplex analysis, i.e., assays must be performed in a series. Addressing these concerns represents a largely unmet research need but more comprehensive parallel analysis of myofibrillar proteins could advance knowledge regarding age- and activity-dependent changes in human muscle. We report a label-free, semi-automated and time efficient LC-MS proteomic workflow for phenotyping the myofibrillar proteome. Application of this workflow in old and young as well as trained and untrained human skeletal muscle yielded several novel observations that were subsequently verified by multiple reaction monitoring (MRM).We report novel data demonstrating that human ageing is associated with lesser myosin light chain 1 content and greater myosin light chain 3 content, consistent with an age-related reduction in type II muscle fibers. We also disambiguate conflicting data regarding myosin regulatory light chain, revealing that age-related changes in this protein more closely reflect physical activity status than ageing per se. This finding reinforces the need to control for physical activity levels when investigating the natural process of ageing. Taken together, our data confirm and extend knowledge regarding age- and activity-related phenotypes. In addition, the MRM transitions described here provide a methodological platform that can be fine-tuned to suite multiple research needs and thus advance myofibrillar phenotyping

Proteomic analysis reveals perturbed energy metabolism and elevated oxidative stress in hearts of rats with inborn low aerobic capacity

Selection on running capacity has created rat phenotypes of high‐capacity runners (HCRs) that have enhanced cardiac function and low‐capacity runners (LCRs) that exhibit risk factors of metabolic syndrome. We analysed hearts of HCRs and LCRs from generation 22 of selection using DIGE and identified proteins from MS database searches. The running capacity of HCRs was six‐fold greater than LCRs. DIGE resolved 957 spots and proteins were unambiguously identified in 369 spots. Protein expression profiling detected 67 statistically significant ( p <0.05; false discovery rate <10%, calculated using q ‐values) differences between HCRs and LCRs. Hearts of HCR rats exhibited robust increases in the abundance of each enzyme of the β‐oxidation pathway. In contrast, LCR hearts were characterised by the modulation of enzymes associated with ketone body or amino acid metabolism. LCRs also exhibited enhanced expression of antioxidant enzymes such as catalase and greater phosphorylation of α B‐crystallin at serine 59, which is a common point of convergence in cardiac stress signalling. Thus, proteomic analysis revealed selection on low running capacity is associated with perturbations in cardiac energy metabolism and provided the first evidence that the LCR cardiac proteome is exposed to greater oxidative stress.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86916/1/3369_ftp.pd

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

Effects of an active warm-up on variation in bench press and back squat (upper and lower body measures).

The present study investigated the magnitude of diurnal variation in back squat and bench press using the MuscleLab linear encoder over three different loads and assessed the benefit of an active warm-up to establish whether diurnal variation could be negated. Ten resistance-trained males underwent (mean ± SD: age 21.0 ± 1.3 years, height 1.77 ± 0.06 m, and body mass 82.8 ± 14.9 kg) three sessions. These included control morning (M, 07:30 h) and evening (E, 17:30 h) sessions (5-min standardized warm-up at 150 W, on a cycle ergometer), and one further session consisting of an extended active warm-up morning trial (ME, 07:30 h) until rectal temperature (Trec) reached previously recorded resting evening levels (at 150 W, on a cycle ergometer). All sessions included handgrip, followed by a defined program of bench press (at 20, 40, and 60 kg) and back squat (at 30, 50, and 70 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. Values for Trec were higher in the E session compared to values in the M session (Δ0.53 °C, P  0.05) to increase from M to E levels. Therefore, MuscleLab linear encoder could detect meaningful differences between the morning and evening for all variables. However, the diurnal variation in bench press and back squat (measures of lower and upper body force and power output) is not explained by time-of-day oscillations in Trec

Controlling rectal and muscle temperatures: Can we offset diurnal variation in repeated sprint performance?

The present study investigated whether increasing morning rectal temperatures (Trec) to resting.evening levels, or decreasing evening Trec or muscle (Tm) temperatures to morning values, would influence repeated sprint (RS) performance in a causal manner. Twelve trained males underwent five sessions [age (mean ± SD) 21.8 ± 2.6 yr, peak oxygen uptake ( peak) 60.6 ± 4.6 mL kg min−1, stature 1.78 ± 0.07 m and body mass 76.0 ± 6.3 kg]. These included a control morning (M, 07:30 h) and evening (E, 17:30 h) session (5-min warm-up), and three further sessions consisting of a warm-up morning trial (ME, on a motorised treadmill) until Trec reached evening levels; and two cool-down evening trials (in 16–17°C water) until Trec (EMrec) or Tm (EMmuscle) values reached morning temperatures, respectively. All sessions included a 3 × 3-s task-specific warm-up followed by 10 × 3-s RS with 30-s recoveries performed on a non-motorised treadmill. Trec and Tm measurements were taken at the start of the protocol and following the warm-up or cool-down period. Values for Trec and Tm were higher in the evening compared to morning values (0.45°C and 0.57°C, P < 0.05). RS performance was lower in the M for distance covered (DC), average power (AP) and average velocity (AV) (9–10%, P < 0.05). Pre-cooling Trec and Tm in the evening reduced RS performance to levels observed in the morning (P < 0.05). However, an active warm-up resulted in no changes in morning RS performance. Diurnal variation in Trec and Tm is not wholly accountable for time-of-day oscillations in RS performance on a non-motorised treadmill; the exact mechanism(s) for a causal link between central temperature and human performance are still unclear and require more research

The combination of smoking with vitamin D deficiency impairs skeletal muscle fiber hypertrophy in response to overload in mice.

Vitamin D deficiency, which is highly prevalent in the general population, exerts similar deleterious effects on skeletal muscles to those induced by cigarette smoking. We examined whether cigarette smoke (CS) exposure and/or vitamin D deficiency impairs the skeletal muscle hypertrophic response to overload. Male C57Bl/6JolaH mice on a normal or vitamin D-deficient diet were exposed to CS or room air for 18 wk. Six weeks after initiation of smoke or air exposure, sham surgery or denervation of the agonists of the left plantaris muscle was performed. The right leg served as internal control. Twelve weeks later, the hypertrophic response was assessed. CS exposure instigated loss of body and muscle mass, and increased lung inflammatory cell infiltration (P P = 0.03). In situ fatigue resistance was elevated in hypertrophied plantaris, irrespective of vitamin D deficiency and/or CS exposure. In conclusion, our data show that CS exposure or vitamin D deficiency alone did not attenuate the hypertrophic response of overloaded plantaris muscles, but this hypertrophic response was weakened when both conditions were combined. These data suggest that current smokers who also present with vitamin D deficiency may be less likely to respond to a training program.NEW & NOTEWORTHY Plantaris hypertrophy caused by compensatory overload after denervation of the soleus and gastrocnemius muscles showed increased mass and fiber dimensions, but to a lesser extent when vitamin D deficiency was combined with cigarette smoking. Fatigue resistance was elevated in hypertrophied plantaris, irrespective of diet or smoking, whereas physical fitness, hypertrophic markers, and in situ plantaris force were similar. These data showed that the hypertrophic response to overload is attenuated when both conditions are combined

Effects of two nights partial sleep deprivation on an evening submaximal weightlifting performance; are 1 h powernaps useful on the day of competition?

We have investigated the effects that sleep restriction (3-h sleep during two consecutive nights) have on an evening (17:00 h) submaximal weightlifting session; and whether this performance improves following a 1-h post-lunch powernap. Fifteen resistance-trained males participated in this study. Before the experimental protocol commenced, 1RM bench press and inclined leg press and normative habitual sleep were recorded. Participants were familiarised with the testing protocol, then completed three experimental conditions with two nights of prescribed sleep: (i) Normal (N): retire at 23:00 h and wake at 06:30 h, (ii) partial sleep-deprivation (SD): retire at 03:30 h and wake at 06:30 h and (iii) partial sleep-deprivation with nap (SDN): retire at 03:30 h and wake at 06:30 h with a 1-h nap at 13:00 h. Each condition was separated by at least 7 days and the order of administration was randomised and counterbalanced. Rectal (Trec) and mean skin (Ts) temperatures, Profile of Mood Scores, subjective tiredness, alertness and sleepiness values were measured at 08:00, 11:00, 14:00 and 17:00 h on the day of the weightlifting session. Following the final temperature measurements at 17:00 h, participants completed a 5-min active warm-up before a ‘strength’ protocol. Participants performed three repetitions of right-hand grip strength, then three repetitions at each incremental load (40%, 60% and 80% of 1RM) for bench press and inclined leg press, with a 5-min recovery in between each repetition. A linear encoder was attached perpendicular to the movement, to the bar used for the exercises. Average power (AP), average force (AF), peak velocity (PV), distance (D) and time-to-peak velocity (tPV) were measured (MuscleLab software) during the concentric phase of the movements for each lift. Data were analysed using general linear models with repeated measures. The main findings were that SD reduced maximal grip (2.7%), bench press (11.2% AP, 3.3% AF and 9.4% PV) and leg press submaximal values (5.7% AP) with a trend for a reduction in AF (3.3% P = 0.06). Furthermore, RPE increased for measures of grip strength, leg and bench press during SD. Following a 1-h powernap (SDN), values of grip and bench press improved to values similar in N, as did tiredness, alertness and sleepiness. There was a main effect for “load” on the bar for both bench and leg press where AP, AF, tPV values increased with load (P < 0.05) and PV decreased from the lightest to the heaviest load for both bench and leg press. An interaction of “load and condition” was present in leg press only, where the rate of change of AP is greater in the N than SD and SDN conditions. In addition, for PV and tPV the rate of change was greater for SDN than N or SD condition values. In summary, SD had a negative effect on grip strength and some components of bench and inclined leg press. The use of a 1-h power nap that ended 3 h before the “strength” assessment had a positive effect on weightlifting performance, subjective mood and ratings of tiredness

Is the diurnal variation in muscle force output detected/detectable when multi-joint movements are analysed using the musclelab force-velocity encoder?

We have investigated the magnitude of diurnal variation in back squat and bench press performance using the MuscleLab force velocity transducer. Thirty resistance-trained males (mean ± SD: age 21.7 ± 1.4 years; body mass 80.5 ± 4.5 kg; height 1.79 ± 0.06 m) underwent two sessions at different times of day: morning (M, 07:30 h) and evening (E, 17:30 h). Each session included a period when rectal temperature (Trec) was measured at rest, a 5-min standardized 150 W warm-up on a cycle ergometer, then defined programme of bench press (at 20, 40 and 60 kg) and back squat (at 30, 50 and 70 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. Values for Trec at rest were higher in the evening compared to morning values (0.48°C, P < 0.0005). Daily variations were apparent for both bench press and back squat performance for AF (1.9 and 2.5%), PV (8.3 and 12.7%) and tPV (-16.6 and -9.8%; where a negative number indicates a decrease in the variable from morning to evening). There was a main effect for load where AF and tPV increased and PV decreased from the lightest load to the heaviest for both bench press and back squat (47.1 and 80.2%; 31.7 and 57.7%; -42.1 and -73.9%; P < 0.0005 where a negative number indicates a decrease in the variable with increasing load). An interaction was found only for tPV, such that the tPV occurs earlier in the evening than the morning at the highest loads (60 and 70 kg) for both bench press and back squat, respectively (mean difference of 0.32 and 0.62 s). In summary, diurnal variation in back squat and bench press was shown; and the tPV in complex multi-joint movements occurs earlier during the concentric phase of exercise when back squat or bench press is performed in the evening compared to the morning. This difference can be detected using a low cost, portable and widely available commercial instrument and enables translation of past laboratory/tightly controlled experimental research in to main-stream coaching practice

Global proteome changes in the rat diaphragm induced by endurance exercise training

Mechanical ventilation (MV) is a life-saving intervention for many critically ill patients. Unfor- tunately, prolonged MV results in the rapid development of diaphragmatic atrophy and weakness. Importantly, endurance exercise training results in a diaphragmatic phenotype that is protected against ventilator-induced diaphragmatic atrophy and weakness. The mechanisms responsible for this exercise-induced protection against ventilator-induced dia- phragmatic atrophy remain unknown. Therefore, to investigate exercise-induced changes in diaphragm muscle proteins, we compared the diaphragmatic proteome from sedentary and exercise-trained rats. Specifically, using label-free liquid chromatography-mass spectrome- try, we performed a proteomics analysis of both soluble proteins and mitochondrial proteins isolated from diaphragm muscle. The total number of diaphragm proteins profiled in the sol- uble protein fraction and mitochondrial protein fraction were 813 and 732, respectively. Endurance exercise training significantly (P<0.05, FDR <10%) altered the abundance of 70 proteins in the soluble diaphragm proteome and 25 proteins of the mitochondrial proteome. In particular, key cytoprotective proteins that increased in relative abundance following exer- cise training included mitochondrial fission process 1 (Mtfp1; MTP18), 3-mercaptopyruvate sulfurtransferase (3MPST), microsomal glutathione S-transferase 3 (Mgst3; GST-III), and heat shock protein 70 kDa protein 1A/1B (HSP70). While these proteins are known to be cytoprotective in several cell types, the cyto-protective roles of these proteins have yet to be fully elucidated in diaphragm muscle fibers. Based upon these important findings, future experiments can now determine which of these diaphragmatic proteins are sufficient and/or required to promote exercise-induced protection against inactivity-induced muscle atrophy

Label‐free profiling of skeletal muscle using high‐definition mass spectrometry

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/109264/1/pmic7807.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/109264/2/pmic7807-sup-0002-FigureS2.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/109264/3/pmic7807-sup-0001-FigureS1.pd