Timothy Etheridge

Introduction: Ageing remains a highly elusive process, with its associated physical and molecular decline continuing to place financial strain on UK healthcare systems. Mitochondrial function is known to play an important role in the ageing phenotype, where it has gained attention as an attractive therapeutic target in attempts to alleviate these pathologies. The current work presents a panel of novel H2S compounds and their effects on C. elegans survival.

Methods: Using the model organism C. elegans we tested dosing of five compounds across the life-course (1μM, 100nM and 10nM, with respective controls for both the compound (i.e. cellular targeting motif alone) and DMSO alone. with age-synchronysed animals exposed from L1 larval stage. Survival assays were performed using a high-throughput microfluidic platform and scored every day for being alive or dead. To infer a putative prole of mitochondrial preservation in any compound-induced lifespan extension, transgenic animals expressing mitochondrial green fluorescent protein (for RT163 only) were also examined for mitochondrial structure across the life-course.

Results: all mitochondrial H2S donors extended C. elegans lifespan. RT163 showed the most prominent effects at a dose of 100nM, with 100 nM also proving an efficacious dose in both RTC1 and RTA302. The endoplasmic reticulum targeting drug, RTER88, failed to extended lifespan versus control conditions. Mitochondrial structure was also assessed in animals exposed to 100nM of RT163 against 100nM of the compound control and DMSO. RT163 was able to preserve networking up to 6 days post-adulthood, highlighting the improved survival is likely mitochondrially mediated.

Conclusion: Mitochondria play an integral role in the ageing process, with strategies to augment its age-dependent extending lifespan. Here we report that H2S targeted to mitochondria increases survival proportions, with some insight into preservation of mitochondrial structure potentially the causal role. Importantly, these effects are seen at orders of magnitude lower than traditional sulfide donors, emphasising the potential that these compounds have to be clinically relevant drugs.

Biology experiments in space seek to increase our understanding of what happens to life beyond Earth and how we can safely send life beyond Earth. Spaceflight is associated with many (mal)adaptations in physiology, including decline in musculoskeletal, cardiovascular, vestibular, and immune systems. Biological experiments in space are inherently challenging to implement. Development of hardware and validation of experimental conditions are critical to ensure the collection of high-quality data. The model organism
. Caenorhabditis elegans
. has been studied in space for more than 20 years to better understand spaceflight-induced (patho)physiology, particularly spaceflight-induced muscle decline. These experiments have used a variety of hardware configurations. Despite this, hardware used in the past was not available for our most recent experiment, the Molecular Muscle Experiment (MME). Therefore, we had to design and validate flight hardware for MME. MME provides a contemporary example of many of the challenges faced by researchers conducting
. C. elegans
. experiments onboard the International Space Station. Here, we describe the hardware selection and validation, in addition to the ground-based experiment scientific validation testing. These experiences and operational solutions allow others to replicate and/or improve our experimental design on future missions.

Resistance exercise (RE) remains a primary approach for minimising aging muscle decline. Understanding muscle adaptation to individual contractile components of RE (eccentric, concentric) might optimise RE-based intervention strategies. Herein, we employed a network-driven pipeline to identify putative molecular drivers of muscle aging and contraction mode responses. RNA-sequencing data was generated from young (21±1 y) and older (70±1 y) human skeletal muscle before and following acute unilateral concentric and contralateral eccentric contractions. Application of weighted gene co-expression network analysis identified 33 distinct gene clusters ('modules') with an expression profile regulated by aging, contraction and/or linked to muscle strength. These included two contraction 'responsive' modules (related to 'cell adhesion' and 'transcription factor' processes) that also correlated with the magnitude of post-exercise muscle strength decline. Module searches for 'hub' genes and enriched transcription factor binding sites established a refined set of candidate module-regulatory molecules (536 hub genes and 60 transcription factors) as possible contributors to muscle aging and/or contraction responses. Thus, network-driven analysis can identify new molecular candidates of functional relevance to muscle aging and contraction mode adaptations.

Mitochondrial dysfunction impairs muscle health and causes subsequent muscle wasting. This study explores the role of mitochondrial dysfunction as an intramuscular signal for the extracellular matrix (ECM)-based proteolysis and, consequentially, muscle cell dystrophy. We found that inhibition of the mitochondrial electron transport chain causes paralysis as well as muscle structural damage in the nematode Caenorhabditis elegans. This was associated with a significant decline in collagen content. Both paralysis and muscle damage could be rescued with collagen IV overexpression, matrix metalloproteinase (MMP), and Furin inhibitors in Antimycin A-treated animal as well as in the C. elegans Duchenne muscular dystrophy model. Additionally, muscle cytosolic calcium increased in the Antimycin A-treated worms, and its down-regulation rescued the muscle damage, suggesting that calcium overload acts as one of the early triggers and activates Furin and MMPs for collagen degradation. In conclusion, we have established ECM degradation as an important pathway of muscle damage.-Sudevan, S. Takiura, M. Kubota, Y. Higashitani, N. Cooke, M. Ellwood, R. A. Etheridge, T. Szewczyk, N. J. Higashitani, A. Mitochondrial dysfunction causes Ca2+ overload and ECM degradation-mediated muscle damage in C. elegans.

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.

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 to 1) muscle mass/protein metabolism: leucine, hydroxyl β-methylbutyrate, creatine, vitamin-D, ursolic acid, and phosphatidic acid; and 2) exercise performance: (i.e. strength or endurance capacity): hydroxyl β-methylbutyrate, carnitine, creatine, nitrates, and β-alanine.

The integrin-adhesome network, which contains >150 proteins, is mechano-transducing and located at discreet positions along the cell-cell and cell-extracellular matrix interface. A small subset of the integrin-adhesome is known to maintain normal muscle morphology. However, the importance of the entire adhesome for muscle structure and function is unknown. We used RNA interference to knock down 113 putative Caenorhabditis elegans homologs constituting most of the mammalian adhesome and 48 proteins known to localize to attachment sites in C. elegans muscle. In both cases, we found >90% of components were required for normal muscle mitochondrial structure and/or proteostasis vs. empty vector controls. Approximately half of these, mainly proteins that physically interact with each other, were also required for normal sarcomere and/or adhesome structure. Next we confirmed that the dystrophy observed in adhesome mutants associates with impaired maximal mitochondrial ATP production (P < 0.01), as well as reduced probability distribution of muscle movement forces compared with wild-type animals. Our results show that the integrin-adhesome network as a whole is required for maintaining both muscle structure and function and extend the current understanding of the full complexities of the functional adhesome in vivo.

Maintenance of skeletal muscle mass is contingent upon the dynamic equilibrium (fasted losses-fed gains) in protein turnover. of all nutrients, the single amino acid leucine (Leu) possesses the most marked anabolic characteristics in acting as a trigger element for the initiation of protein synthesis. While the mechanisms by which Leu is 'sensed' have been the subject of great scrutiny, as a branched-chain amino acid, Leu can be catabolized within muscle, thus posing the possibility that metabolites of Leu could be involved in mediating the anabolic effect(s) of Leu. Our objective was to measure muscle protein anabolism in response to Leu and its metabolite HMB. Using [1,2-(13)C2]Leu and [(2)H5]phenylalanine tracers, and GC-MS/GC-C-IRMS we studied the effect of HMB or Leu alone on MPS (by tracer incorporation into myofibrils), and for HMB we also measured muscle proteolysis (by arteriovenous (A-V) dilution). Orally consumed 3.42 g free-acid (FA-HMB) HMB (providing 2.42 g of pure HMB) exhibited rapid bioavailability in plasma and muscle and, similarly to 3.42 g Leu, stimulated muscle protein synthesis (MPS; HMB +70% vs. Leu +110%). While HMB and Leu both increased anabolic signalling (mechanistic target of rapamycin; mTOR), this was more pronounced with Leu (i.e. p70S6K1 signalling 90 min vs. 30 min for HMB). HMB consumption also attenuated muscle protein breakdown (MPB; -57%) in an insulin-independent manner. We conclude that exogenous HMB induces acute muscle anabolism (increased MPS and reduced MPB) albeit perhaps via distinct, and/or additional mechanism(s) to Leu.

Chronic reductions in tissue O2 tension (hypoxia) are associated with muscle atrophy and blunted hypertrophic responses to resistance exercise (RE) training. However, the effect of hypoxia on muscle protein synthesis (MPS) at rest and after RE is unknown. In a crossover study, seven healthy men (21.4 ± 0.7 yr) performed unilateral leg RE (6 × 8 repetitions at 70% 1-repetition maximum) under normoxic (20.9% inspired O2) and normobaric hypoxic (12% inspired O2 for 3.5 h) postabsorptive conditions. Immediately after RE the rested leg was biopsied, and a primed continuous infusion of [1,2-13C2]leucine was maintained for 2.5 h before final biopsies from both legs to measure tracer incorporation and signaling responses (i.e. ribosomal S6 kinase 1). After 3.5 h of hypoxia, MPS was not different from normoxia in the rested leg (normoxia 0.033 ± 0.016 vs. hypoxia 0.043 ± 0.016%/h). MPS increased significantly from baseline 2.5 h after RE in normoxia (0.033 ± 0.016 vs. 0.104 ± 0.038%/h) but not hypoxia (0.043 ± 0.016 vs. 0.060 ± 0.063%/h). A significant linear relationship existed between MPS 2.5 h after RE in hypoxia and mean arterial blood O2 saturation during hypoxia ( r2 = 0.49, P = 0.04). Phosphorylation of p70S6KThr389 remained unchanged in hypoxia at rest but increased after RE in both normoxia and hypoxia (2.6 ± 1.2-fold and 3.4 ± 1.1-fold, respectively). Concentrations of the hypoxia-responsive mTOR inhibitor regulated in development and DNA damage-1 were unaltered by hypoxia or RE. We conclude that normobaric hypoxia does not reduce MPS over 3.5 h at rest but blunts the increased MPS response to acute RE to a degree dependent on extant SpO2.

. The ultimate survival of humanity is dependent upon colonization of other planetary bodies. Key challenges to such habitation are (patho)physiologic changes induced by known, and unknown, factors associated with long-duration and distance space exploration. However, we currently lack biological models for detecting and studying these changes. Here, we use a remote automated culture system to successfully grow an animal in low Earth orbit for six months. Our observations, over 12 generations, demonstrate that the multi-cellular soil worm
. Caenorhabditis elegans
. develops from egg to adulthood and produces progeny with identical timings in space as on the Earth. Additionally, these animals display normal rates of movement when fully fed, comparable declines in movement when starved, and appropriate growth arrest upon starvation and recovery upon re-feeding. These observations establish
. C. elegans
. as a biological model that can be used to detect changes in animal growth, development, reproduction and behaviour in response to environmental conditions during long-duration spaceflight. This experimental system is ready to be incorporated on future, unmanned interplanetary missions and could be used to study cost-effectively the effects of such missions on these biological processes and the efficacy of new life support systems and radiation shielding technologies.
.

Recently we demonstrated that the effectiveness of RNAi interference (RNAi) for inhibiting gene expression is maintained during spaceflight in the worm Caenorhabditis elegans and argued for the biomedical importance of this finding. We also successfully utilized green fluorescent protein (GFP)-tagged proteins to monitor changes in GPF localization during flight. Here we discuss potential applications of RNAi and GFP in spaceflight studies and the ramifications of these experiments for the future of space life-sciences research.

The essential amino acids (EAA) activate anabolic signalling through mechanisms, which are unclear in detail but include increased signalling through the mammalian target of rapamycin complex 1 (mTORC1). of all the EAA, the branched chain amino acid (BCAA) leucine has been suggested as the most potent in stimulating protein synthesis, although there have been no studies investigating the effects of each EAA on anabolic signalling pathways. We therefore undertook a systematic analysis of the effect of each EAA on mTORC1 signalling in C2C12 myotubes whereby cells were serum (4 h) and amino acid (1 h) starved before stimulation with 2 mM of each amino acid. Immunoblotting was used to detect phosphorylated forms of protein kinase B (Akt)/mTORC1 signalling enzymes. The phosphorylation of Akt was unchanged by incubation with EAA. Phosphorylation of mTOR and 4E binding protein-1 (4EBP1) were increased 1.67 +/- 0.1-fold and 2.5 +/- 0.1-fold, respectively, in response to leucine stimulation but not in response to any other EAA. The phosphorylation of ribosomal s6 kinase (p70S6K1) was increased by stimulation with all EAA with the exceptions of isoleucine and valine. However, the increase with leucine was significantly greater, 5.9 +/- 0.3-fold compared to 1.6-2.0-fold for the non-BCAA EAA. This pattern of activation was identical in ribosomal protein s6 (RPS6) with the additional effect of leucine being 3.8 +/- 0.3-fold versus 1.5-2.0-fold. Phosphorylation of eukaryotic initiation/elongation factors eIF2alpha and eEF2 were unaffected by EAA. We conclude that leucine is unique amongst the amino acids in its capacity to stimulate both mTOR and 4EBP1 phosphorylation and to enhance p70S6K1 signalling.

The purpose of this study was to examine the effects of acute protein ingestion on the recovery of muscle function and markers of muscle damage in the 72 h post eccentric-exercise. Nine recreationally active males recorded quadriceps maximum isometric voluntary contraction (MVC), peak 5 s power output (PPO), and perceived muscle soreness. Plasma creatine kinase (CK) and protein carbonyl (PC) content were measured prior to exercise. Delayed-onset muscle soreness (DOMS) was induced by a 30 min downhill run (-10 degrees ) at a target intensity of 75% age-predicted heart rate maximum, immediately followed by ingestion of 100 g protein (containing 40 g essential amino acids; PRO) or placebo (CON) solution. The pre-exercise measures were re-taken in the subsequent 24, 48, and 72 h. CK, PC, and perceived muscle soreness increased significantly following exercise and with each supplement at 24 h. PC and muscle soreness remained elevated at 48 and 72 h (p < 0.05), whereas CK returned to baseline values. No difference between conditions was observed for these measures. Peak MVC significantly declined in CON to -7.9% at 24 h, reaching a nadir of -10% at 48 h (p < 0.05). In the PRO group, MVC remained within pre-exercise values at all time points. PPO followed a similar trend, reaching its nadir of -8.7% at 48 h in CON (p < 0.05), but had recovered in the PRO trial. Ingestion of a single post-exercise protein mixture increases the rate of force and power restoration at 48 h, suggesting potential for protein as an ergogenic aid during the DOMS period.