Exploring metabolic and molecular mechanisms regulating age-related declines in human skeletal muscle regenerative capacity.

The underlying mechanisms regulating the ability of skeletal muscle to regenerate after acute “damaging” eccentric or “non-damaging” concentric exercise in young human skeletal muscle is poorly defined. Age-related impairments in the regenerative mechanisms may contribute to the age-related loss of muscle mass and function, which has negative consequences for overall health and disease. Thus, the first aim of this thesis was to initially investigate multiple targeted mechanisms previously implicated in the regeneration process, over a comprehensive time-course following eccentric versus concentric exercise in young adults. Within this study it was found that post-exercise, in general, increased anabolic and repressed catabolic signalling preceded functional decline, whereas inflammation and ubiquitin proteasome system-related breakdown increased once functional recovery was initiated/achieved. Eccentric exercise led to greater anabolic signalling and inflammatory signalling response. As such, this study has provided a benchmark of muscle regeneration in young skeletal muscle, which implicates early anabolic and catabolic regulation in the rapid adaptation of muscle, whereas inflammation and ubiquitin proteasome system-related breakdown likely mediate longer term remodelling/adaptations, which may be greater following eccentric exercise. Using this benchmark, the aim of the second study was to identify age-related changes in targeted regenerative mechanisms. Concentric exercise did not cause a molecular regenerative response, whilst eccentric exercise induced anabolic signalling and satellite cell activation, prior to and at the nadir of force, respectively. Compared to the younger adults, ageing per se was associated with increased inflammation, whilst anabolic and catabolic signalling post-eccentric and concentric exercise was blunted. Interestingly, satellite cell activity was induced in the old only following eccentric exercise. These data suggest that eccentric exercise is potentially more advantageous for promoting muscle growth versus concentric exercise in older adults. Whilst, compared to the young, the old displayed blunted molecular responses which might underlie blunted muscle growth during ageing. Furthermore, the activation of satellite cells in the old might be the result of the impaired molecular mechanisms being suboptimal for repair thus, requiring additional regenerative means. In order to further characterise ageing muscle and the mechanisms of muscle regeneration, RNA sequencing was performed at the time of peak anabolic signalling to highlight more global and novel molecular networks. Ageing per se revealed genes involved in blood vessel development, plasma membrane and cell-cell junction expression were down-regulated, thus implicating these processes in age-related muscle loss. Following concentric exercise in older adults, there was an up-regulation of structural transcripts whilst there was a general down-regulation of genes related to metabolism, which might suggest impaired metabolism post-concentric exercise. Perhaps the blunted transcript responses contribute to the often observed age-related blunting of muscle mass adaptations in response to exercise training. Collectively, the data from this thesis has important implications for developing interventions for maximising hypertrophic responses and for counteracting the suboptimal regenerative responses observed in older adults

Proteomic features of skeletal muscle adaptation to resistance exercise training as a function of age

YesResistance exercise training (RET) can counteract negative features of muscle ageing but older age associates with reduced adaptive capacity to RET. Altered muscle protein networks likely contribute to ageing RET adaptation; therefore, associated proteome-wide responses warrant exploration. We employed quantitative sarcoplasmic proteomics to compare age-related proteome and phosphoproteome responses to RET. Thigh muscle biopsies were collected from eight young (25 ± 1.1 years) and eight older (67.5 ± 2.6 years) adults before and after 20 weeks supervised RET. Muscle sarcoplasmic fractions were pooled for each condition and analysed using Isobaric Tags for Relative and Absolute Quantification (iTRAQ) labelling, tandem mass spectrometry and network-based hub protein identification. Older adults displayed impaired RET-induced adaptations in whole-body lean mass, body fat percentage and thigh lean mass (P > 0.05). iTRAQ identified 73 differentially expressed proteins with age and/or RET. Despite possible proteomic stochasticity, RET improved ageing profiles for mitochondrial function and glucose metabolism (top hub; PYK (pyruvate kinase)) but failed to correct altered ageing expression of cytoskeletal proteins (top hub; YWHAZ (14-3-3 protein zeta/delta)). These ageing RET proteomic profiles were generally unchanged or oppositely regulated post-RET in younger muscle. Similarly, RET corrected expression of 10 phosphoproteins altered in ageing, but these responses were again different vs. younger adults. Older muscle is characterised by RET-induced metabolic protein profiles that, whilst not present in younger muscle, improve untrained age-related proteomic deficits. Combined with impaired cytoskeletal adhesion responses, these results provide a proteomic framework for understanding and optimising ageing muscle RET adaptation.TE was supported by a postdoctoral fellowship from the Japan Society for the Promotion of Science and the Royal Society (JSPS/FF1/435). This work was supported by grants from the Medical Research Council (MR/T026014/1 and G0801271) and the Biotechnology and Biological Sciences Research Council (BB/X510697/1 and BB/C516779/1)

Muscle strength deficiency and mitochondrial dysfunction in a muscular dystrophy model of C. elegans and its functional response to drugs

Muscle strength is a key clinical parameter used to monitor the progression of human muscular dystrophies, including Duchenne and Becker muscular dystrophies. Although Caenorhabditis elegans is an established genetic model for studying the mechanisms and treatments of muscular dystrophies, analogous strength-based measurements in this disease model are lacking. Here, we describe the first demonstration of the direct measurement of muscular strength in dystrophin-deficient C. elegans mutants using a micropillar-based force measurement system called NemaFlex. We show that dys-1(eg33) mutants, but not dys-1(cx18) mutants, are significantly weaker than their wild-type counterparts in early adulthood, cannot thrash in liquid at wild-type rates, display mitochondrial network fragmentation in the body wall muscles, and have an abnormally high baseline mitochondrial respiration. Furthermore, treatment with prednisone, the standard treatment for muscular dystrophy in humans, and melatonin both improve muscular strength, thrashing rate and mitochondrial network integrity in dys-1(eg33), and prednisone treatment also returns baseline respiration to normal levels. Thus, our results demonstrate that the dys-1(eg33) strain is more clinically relevant than dys-1(cx18) for muscular dystrophy studies in C. elegans. This finding, in combination with the novel NemaFlex platform, can be used as an efficient workflow for identifying candidate compounds that can improve strength in the C. elegans muscular dystrophy model. Our study also lays the foundation for further probing of the mechanism of muscle function loss in dystrophin-deficient C. elegans, leading to knowledge translatable to human muscular dystrophy

"Nutraceuticals" in relation to human skeletal muscle and exercise.

Skeletal muscles have a fundamental role in locomotion and whole body metabolism, with muscle mass and quality being linked to improved health and even lifespan. Optimizing nutrition in combination with exercise is considered an established, effective ergogenic practice for athletic performance. Importantly, exercise and nutritional approaches also remain arguably the most effective countermeasure for muscle dysfunction associated with aging and numerous clinical conditions, e.g., cancer cachexia, COPD, and organ failure, via engendering favorable adaptations such as increased muscle mass and oxidative capacity. Therefore, it is important to consider the effects of established and novel effectors of muscle mass, function, and metabolism in relation to nutrition and exercise. To address this gap, in this review, we detail existing evidence surrounding the efficacy of a nonexhaustive list of macronutrient, micronutrient, and "nutraceutical" compounds alone and in combination with exercise in relation to skeletal muscle mass, metabolism (protein and fuel), and exercise performance (i.e., strength and endurance capacity). It has long been established that macronutrients have specific roles and impact upon protein metabolism and exercise performance, (i.e., protein positively influences muscle mass and protein metabolism), whereas carbohydrate and fat intakes can influence fuel metabolism and exercise performance. Regarding novel nutraceuticals, we show that the following ones in particular may have effects in relation to1) muscle mass/protein metabolism: leucine, hydroxyl β-methylbutyrate, creatine, vitamin-D, ursolic acid, and phosphatidic acid; and2) exercise performance: (i.e., strength or endurance capacity): hydroxyl β-methylbutyrate, carnitine, creatine, nitrates, and β-alanine

Worms in Space for Outreach on Earth:Space Life Science Activities for the Classroom

Long term spaceflight is associated with the loss of skeletal muscle mass and function. The Molecular Muscle Experiment (MME) seeks to identify the causes of muscle decline in space and test potential therapies to attenuate this in the microscopic worm,C. elegans. This is the first UK-led experiment in the almost two-decade history of the International Space Station. We therefore intend to complete significant and widespread educational outreach activities to promote interest in science, technology, engineering and maths (STEM), and to increase engagement with our space life science experiment. This paper describes three education outreach activities relating to our MME experiment that are suitable for use in the classroom, including: (i) observing normal and mutant worms; (ii) observing the effect of unloading (simulation of microgravity); and (iii) handling spaceflight hardware. Activity packs are provided at a ‘starter’ and ‘advanced’ level to support these activities. This paper also provides three posters that may be used as learning resources for educators that give information on: (i) why worms are used for research; (ii) spaceflight human physiology; and (iii) the specifics of our MME. Details of further planned engagement activities are outlined to increase the awareness of the MME

The acute transcriptional response to resistance exercise: impact of age and contraction mode

Optimization of resistance exercise (RE) remains a hotbed of research for muscle building and maintenance. However, the interactions between the contractile components of RE (i.e. concentric (CON) and eccentric (ECC)) and age, are poorly defined. We used transcriptomics to compare age-related molecular responses to acute CON and ECC exercise. Eight young (21±1 y) and eight older (70±1 y) exercise-naïve male volunteers had vastus lateralis biopsies collected at baseline and 5 h post unilateral CON and contralateral ECC exercise. RNA was subjected to next-generation sequencing and differentially expressed (DE) genes tested for pathway enrichment using Gene Ontology (GO). The young transcriptional response to CON and ECC was highly similar and older adults displayed moderate contraction-specific profiles, with no GO enrichment. Age-specific responses to ECC revealed 104 DE genes unique to young, and 170 DE genes in older muscle, with no GO enrichment. Following CON, 15 DE genes were young muscle-specific, whereas older muscle uniquely expressed 147 up-regulated genes enriched for cell adhesion and blood vessel development, and 28 down-regulated genes involved in mitochondrial respiration, amino acid and lipid metabolism. Thus, older age is associated with contraction-specific regulation often without clear functional relevance, perhaps reflecting a degree of stochastic age-related dysregulation.This article is freely available via Open Access. Click on the Publisher URL to access it via the publisher's site.CSD was funded by a doctoral training studentship from Bournemouth University. This work was generously supported by the Wellcome Trust Institutional Strategic Support Award (WT105618MA). RMA is generously supported by the Wellcome Trust Institutional Strategic Support Award (WT105618MA) and an EPSRC/BBSRC Innovation Fellowship (EP/S001352/1). We acknowledge the Medical Research Council [grant number MR/P021220/1] [grant number MR/K00414X/1] and Arthritis Research UK [grant number 19891] as part of the MRC-ARUK Centre for Musculoskeletal Ageing Research awarded to the Universities of Nottingham and Birmingham, and the National Institute for Health Research, Nottingham Biomedical Research Centre. This work was supported by the Biotechnology and Biological Sciences Research Council [grant number BB/N015894/1]. This work was supported by a grant from the Swedish Research Council for Sport Science (dnr 2016/125 and dnr 2017/143). C.R.G.W is supported by the Biotechnology and Biological Sciences Research Council-funded South West Biosciences Doctoral Training Partnership [BB/J014400/1; BB/M009122/1].Published versio

Molecular mechanisms underpinning favourable physiological adaptations to exercise prehabilitation for urological cancer surgery

BACKGROUND: Surgery for urological cancers is associated with high complication rates and survivors commonly experience fatigue, reduced physical ability and quality of life. High-intensity interval training (HIIT) as surgical prehabilitation has been proven effective for improving the cardiorespiratory fitness (CRF) of urological cancer patients, however the mechanistic basis of this favourable adaptation is undefined. Thus, we aimed to assess the mechanisms of physiological responses to HIIT as surgical prehabilitation for urological cancer. METHODS: Nineteen male patients scheduled for major urological surgery were randomised to complete 4-weeks HIIT prehabilitation (71.6 ± 0.75 years, BMI: 27.7 ± 0.9 kg·m 2) or a no-intervention control (71.8 ± 1.1 years, BMI: 26.9 ± 1.3 kg·m 2). Before and after the intervention period, patients underwent m. vastus lateralis biopsies to quantify the impact of HIIT on mitochondrial oxidative phosphorylation (OXPHOS) capacity, cumulative myofibrillar muscle protein synthesis (MPS) and anabolic, catabolic and insulin-related signalling. RESULTS: OXPHOS capacity increased with HIIT, with increased expression of electron transport chain protein complexes (C)-II (p = 0.010) and III (p = 0.045); and a significant correlation between changes in C-I (r = 0.80, p = 0.003), C-IV (r = 0.75, p = 0.008) and C-V (r = 0.61, p = 0.046) and changes in CRF. Neither MPS (1.81 ± 0.12 to 2.04 ± 0.14%·day −1 , p = 0.39) nor anabolic or catabolic proteins were upregulated by HIIT (p > 0.05). There was, however, an increase in phosphorylation of AS160 Thr642 (p = 0.046) post-HIIT. CONCLUSIONS: A HIIT surgical prehabilitation regime, which improved the CRF of urological cancer patients, enhanced capacity for skeletal muscle OXPHOS; offering potential mechanistic explanation for this favourable adaptation. HIIT did not stimulate MPS, synonymous with the observed lack of hypertrophy. Larger trials pairing patient-centred and clinical endpoints with mechanistic investigations are required to determine the broader impacts of HIIT prehabilitation in this cohort, and to inform on future optimisation (i.e., to increase muscle mass)

Comparative Analysis of Muscle Atrophy During Spaceflight, Nutritional Deficiency and Disuse in the Nematode Caenorhabditis elegans

While spaceflight is becoming more common than before, the hazards spaceflight and space microgravity pose to the human body remain relatively unexplored. Astronauts experience muscle atrophy after spaceflight, but the exact reasons for this and solutions are unknown. Here, we take advantage of the nematode C. elegans to understand the effects of space microgravity on worm body wall muscle. We found that space microgravity induces muscle atrophy in C. elegans from two independent spaceflight missions. As a comparison to spaceflight-induced muscle atrophy, we assessed the effects of acute nutritional deprivation and muscle disuse on C. elegans muscle cells. We found that these two factors also induce muscle atrophy in the nematode. Finally, we identified clp-4, which encodes a calpain protease that promotes muscle atrophy. Mutants of clp-4 suppress starvation-induced muscle atrophy. Such comparative analyses of different factors causing muscle atrophy in C. elegans could provide a way to identify novel genetic factors regulating space microgravity-induced muscle atrophy

Space omics research in Europe: Contributions, geographical distribution and ESA member state funding schemes

The European research community, via European Space Agency (ESA) spaceflight opportunities, has significantly contributed toward our current understanding of spaceflight biology. Recent molecular biology experiments include “omic” analysis, which provides a holistic and systems level understanding of the mechanisms underlying phenotypic adaptation. Despite vast interest in, and the immense quantity of biological information gained from space omics research, the knowledge of ESA-related space omics works as a collective remains poorly defined due to the recent exponential application of omics approaches in space and the limited search capabilities of pre-existing records. Thus, a review of such contributions is necessary to clarify and promote the development of space omics among ESA and ESA state members. To address this gap, in this review, we i) identified and summarized omics works led by European researchers, ii) geographically described these omics works, and iii) highlighted potential caveats in complex funding scenarios among ESA member states

The role of resveratrol on skeletal muscle cell differentiation and myotube hypertrophy during glucose restriction

Glucose restriction (GR) impairs muscle cell differentiation and evokes myotube atrophy. Resveratrol treatment in skeletal muscle cells improves inflammatory-induced reductions in skeletal muscle cell differentiation. We therefore hypothesised that resveratrol treatment would improve muscle cell differentiation and myotube hypertrophy in differentiating C2C12 myoblasts and mature myotubes during GR. Glucose restriction at 0.6 g/L (3.3 mM) blocked differentiation and myotube hypertrophy versus high-glucose (4.5 g/L or 25 mM) differentiation media (DM) conditions universally used for myoblast culture. Resveratrol (10 μM) treatment increased SIRT1 phosphorylation in DM conditions, yet did not improve differentiation when administered to differentiating myoblasts in GR conditions. Resveratrol did evoke increases in hypertrophy of mature myotubes under DM conditions with corresponding elevated Igf-I and Myhc7 gene expression, coding for the ‘slow’ type I MYHC protein isoform. Inhibition of SIRT1 via EX-527 administration (100 nM) also reduced myotube diameter and area in DM conditions and resulted in lower gene expression of Myhc 1, 2 and 4 coding for ‘intermediate’ and ‘faster’ IIx, IIa and IIb protein isoforms, respectively. Resveratrol treatment did not appear to modulate phosphorylation of energy-sensing protein AMPK or protein translation initiator P70S6K. Importantly, in mature myotubes, resveratrol treatment was able to ameliorate reduced myotube growth in GR conditions over an acute 24-h period, but not over 48–72 h. Overall, resveratrol evoked myotube hypertrophy in DM conditions while favouring ‘slower’ Myhc gene expression and acutely ameliorated impaired myotube growth observed during glucose restriction