Microgravity provides a unique model for understanding accelerated skeletal muscle loss, and potentially a model of muscle ageing, offering insights into the molecular mechanisms underlying reductions in muscle mass and function. During spaceflight, astronauts experience pronounced skeletal muscle atrophy. These effects appear similar to age-related muscle decline on Earth but on a significantly shorter timescale. Despite the incorporation of daily aerobic and resistance exercise on the International Space Station (ISS), countermeasures remain suboptimal, reflecting analogous challenges in exercise efficacy observed in ageing populations. The MicroAge Mission aimed to exploit microgravity conditions aboard the ISS to determine whether the molecular mechanisms underpinning reduced adaptive responses to contractile activity during ageing are analogous to those induced by spaceflight. The mission also explored proof-of-concept genetic interventions, including overexpression of Heat Shock Protein 10 (HSP10), a mitochondrial chaperone, to mitigate muscle atrophy and functional loss. To conduct these investigations, a tissue-engineering approach was employed to fabricate human skeletal muscle constructs, which were secured to custom-designed 3D-printed scaffolds. The scaffolds featured integrated microfluidic channels designed to interface with the fluid handling system within the flight hardware. The hardware, developed by Kayser Space Ltd, was specifically designed to interface with the European Space Agency’s (ESA) Kubik incubator located within the Columbus module of the ISS. This research addresses critical methodological constraints in low Earth orbit (LEO) experimentation, providing a detailed account of pre-flight protocol development, muscle construct biofabrication techniques, and operational considerations. The findings establish a translational framework for future investigations into musculoskeletal degeneration, with implications for therapeutic strategies targeting both terrestrial ageing and astronaut musculoskeletal health. ### Competing Interest Statement The authors have declared no competing interest. Science and Technology Facilities Council, ST/S003061/1

Substantial reductions in muscle motor unit numbers accompany ageing and occur in parallel the age-related changes in skeletal muscle mass and fibre number. These motor unit changes are reflected in reduced motor neuron numbers and size, axonal integrity and disrupted pre-and post-synaptic neuromuscular junctions (NMJ). Conversely, data indicate that the effects of ageing on neuromuscular transmission are relatively minor. Some authors have therefore argued that structural degeneration of motor axons and NMJ are unimportant in the pathogenesis of sarcopenia and for a non-neurogenic origin for ageing-induced muscle loss. Increased Reactive Oxygen Species (ROS) activities and changes in redox status are a feature of ageing and may play a key role in muscle loss through increased mitochondrial peroxide generation. This article will review the changes in motor units and NMJ seen during ageing and develop the argument that the changes in muscle mitochondrial peroxide generation and redox status may be caused by age-related changes in neuromuscular structure, but are not directly related to neuromuscular transmission. This provides an alternative explanation on how age-related changes in neural tissue might drive skeletal muscle fibre loss and weakness. Exercise interventions are known to reduce muscle loss and weakness in the elderly, but studies of such interventions on age-related changes in motor units, motor neurons or NMJ structure and function provide conflicting data. A further aim is therefore to identify areas where there is a need for novel research to understand whether, and how, targeted or long-term exercise might influence neuromuscular changes in ageing.

Compared with 2D monolayers, 3D models more closely mimic native muscle tissue and allow functional measurements. A more complete understanding of how culture conditions and duration affect myotube maturity/function is crucial for validating the transition to 3D systems. Human skeletal muscle cells were cultured as 2D monolayers or within 3D hydrogels for up to 21 days. Quantitative proteomic analysis and functional measurements were conducted to evaluate muscle cell differentiation. Myoblasts differentiated into myotubes by 8 days in both environments; however, at Day 8, 3D constructs exhibited a predominantly slow-twitch phenotype, compared with the mixed fiber type of 2D monolayers. By Day 21, 3D constructs demonstrated enhanced mitochondrial maturity, extracellular matrix remodeling, and a fast-twitch phenotype, indicated by increased myosin-2 abundance (Log2(FC)>1.29, p <0.05). Passive tension increased by >20% following prolonged culture of 3D muscle constructs, but contractile forces reduced by >40%. This study provides a comprehensive proteomic profile of human skeletal muscle cells in 2D and 3D, demonstrating that 3D culture promoted myotube maturity and highlighting the importance of selecting appropriate culture conditions. Data suggest 8 days of differentiation as ideal for achieving peak contractile force in 3D constructs, providing optimal models for testing interventions aimed at preserving muscle function.

This study delineated the mechanisms of paclitaxel (PTX) assistance in overcoming radioresistance in hepatoma and human lung adenocarcinoma (HLAC) cells.The TUNEL assay was used as an index of radiosensitivity, and the MTT assay assessed the efficacy of various combined PTX/RT treatments. The efficacy of PTX disruptions of hypoxia-inducible factor-1 alpha (HIF-1α) was assessed using Western blotting.Normoxically overexpressed HIF-1α in hepatoma J5 cells was mechanistically linked to activation of the bFGF/PI3K/Akt pathway because the viability of these cells was strongly inhibited by either Akt inhibitors or an HIF-1α inhibitor. All of the cell lines used were extremely sensitive to PTX, and these effects also correlated excellently with HIF-1α suppression. We designed five combined radiation-PTX protocols of varying dose duration and treatment sequences against CL1-1 cells based on the gathered data. Pretreatment of CL1-1 cells with PTX (100 nM) for 24 h before irradiation (2.5 Gy) was the best combined protocol to achieve maximum radiosensitizing effects.Our data clearly indicate that PTX pretreatment is an effective radiosensitizing procedure against HIF-1α-expressing hepatoma and HLAC cells, which are constitutively endowed with radioresistance.

Skeletal muscle adaptation to contractile activity is modulated by redox signalling, primarily through reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). Early research framed ROS as deleterious byproducts of exercise, but subsequent studies have established their roles as signalling molecules involved in mitochondrial biogenesis, stress responses and metabolic regulation. Central to this process appear to be peroxiredoxins (Prdxs), particularly Prdx2, which current evidence suggests mediate redox relays by sensing physiological H2O2 levels and initiating transcriptional programs. Our recent findings demonstrate that low levels of H2O2, or electrically induced contractions, rapidly oxidise Prdx1, Prdx2 and Prdx3 in mouse muscle fibres. Transcriptomic analysis of human skeletal muscle myotubes confirmed that Prdx2 is essential for upregulating mitochondrial genes in response to H2O2 or contraction. With ageing, skeletal muscle exhibits impaired redox signalling with elevated ROS levels. Using an ageing mouse model, we observed diminished Prdx2 oxidation during contraction, suggesting redox signalling dysfunction. This impaired response likely contributes to sarcopenia by blunting the adaptive capacity of aged muscle. Our findings emphasise the importance of redox homeostasis (not merely ROS suppression) in maintaining muscle health. Understanding the nuanced role of ROS and Prdxs in exercise adaptation and ageing could inform therapeutic strategies aimed at restoring redox-sensitive signalling to preserve muscle function across the lifespan.

Skeletal muscle generates superoxide during contractions, which is converted to hydrogen peroxide (H2O2). H2O2 has been proposed to activate signalling pathways and transcription factors that regulate adaptive responses to exercise, but the concentration required to oxidize and activate key redox-sensitive signalling proteins in vitro is much higher than the typical intracellular levels seen in muscle after exercise. We hypothesized that 2-Cys-peroxiredoxins (PRDX), which rapidly oxidize in the presence of physiological concentrations of H2O2, serve as intermediary signalling molecules and play a crucial role in activating adaptive pathways following muscle contractions. This study has examined the human muscle myotube responses to contractile activity, or exposure to low extracellular concentrations (2.5-5 mu M) of H2O2 and whether knock down of muscle PRDX2 alters the differential gene expression (DEG) that results from these stresses. Exposure of human skeletal muscle myotubes to a 15 min period of aerobic electrically stimulated isometric contractions or 5 mu M H2O2 induced substantial changes in DEG with modification of many genes associated with adaptations of skeletal muscle to contractile activity. Common DEG in these conditions included upregulation of genes associated with increased mitochondrial oxidative phosphorylation, including COX1, COX2, COX3 and ATP6. In myotubes with PRDX2 knock down (94 % decrease in PRDX2 mRNA), the upregulation of genes associated with increased mitochondrial oxidative phosphorylation was abolished following contractile activity or exposure to H2O2. These data indicate that a common effect of contractile activity and exposure to "physiological" levels of H2O2 in human myotubes is to increase the expression of multiple genes associated with increased mitochondrial oxidative phosphorylation. Furthermore, these effects were abolished in PRDX2 knock down myotubes indicating that adaptations to upregulate multiple genes related to increased mitochondrial capacity in human muscle myotubes in response to exercise is both redox regulated and requires PRDX2 as an essential mediator of the effects of H2O2.

Since the first clear observation that skeletal muscle contained increased amounts of free radical species in the early 1980s, there has been increasing interest in how such species are formed and what functions they might have in skeletal muscle. Superoxide and nitric oxide are formed in increased amounts in skeletal muscle during contractile activity and these species together with the important derivative, hydrogen peroxide, are now recognised to play an important role in the physiological responses of muscle to contractile activity or exercise. In this chapter the potential sites for generation of superoxide and hydrogen peroxide during contractile activity will be examined together with the mechanisms for their formation. A major role for these species appears to be to signal the initiation of responses by which the muscle adapts to contractile activity and the potential mechanisms by which they exert such effects will be discussed. This appears to be a still poorly understood area of skeletal muscle biology which however has the potential for exploitation to optimise the beneficial effects of exercise and may well point to pharmacological approaches that can provide the benefits of exercise in immobile, ill or infirm subjects that are unable to take part in traditional exercise regimens.

As a widespread global issue, protein deficiency hinders development and optimal growth in offspring. Maternal low-protein diet influences the development of age-related diseases, including sarcopenia, by altering the epigenome and organ structure through potential increase in oxidative stress. However, the long-term effects of lactational protein restriction or postnatal lifelong protein restriction on the neuromuscular system have yet to be elucidated. Our results demonstrated that feeding a normal protein diet after lactational protein restriction did not have significant impacts on the neuromuscular system in later life. In contrast, a lifelong low-protein diet induced a denervation phenotype and led to demyelination in the sciatic nerve, along with an increase in the number of centralised nuclei and in the gene expression of atrogenes at 18 months of age, indicating an induced skeletal muscle atrophy. These changes were accompanied by an increase in proteasome activity in skeletal muscle, with no significant alterations in oxidative stress or mitochondrial dynamics markers in skeletal muscle later in life. Thus, lifelong protein restriction may induce skeletal muscle atrophy through changes in peripheral nerves and neuromuscular junctions, potentially contributing to the early onset or exaggeration of sarcopenia.

The discovery that contracting skeletal muscle generates reactive oxygen species (ROS) was first reported over 40 years ago. The prevailing view in the 1980s was that exercise-induced ROS production promotes oxidation of proteins and lipids resulting in muscle damage. However, a paradigm shift occurred in the 1990s as growing research revealed that ROS are signaling molecules, capable of activating transcriptional activators/coactivators and promoting exercise-induced muscle adaptation. Growing evidence supports the notion that reduction-oxidation (redox) signaling pathways play an important role in the muscle remodeling that occurs in response to endurance exercise training. This review examines the specific role that redox signaling plays in this endurance exercise-induced skeletal muscle adaptation. We begin with a discussion of the primary sites of ROS production in contracting muscle fibers followed by a summary of the antioxidant enzymes involved in the regulation of ROS levels in the cell. We then discuss which redox-sensitive signaling pathways promote endurance exercise-induced muscle adaptation and debate the strength of the evidence supporting the notion that redox signaling plays an essential role in muscle adaptation to endurance exercise training. In hopes of stimulating future research, we highlight several important unanswered questions in this field.

Reactive oxygen species are generated by multiple mechanisms during contractile activity in exercising skeletal muscle and are recognised to play a role in signaling adaptations to the contractions. The sources of the superoxide and hydrogen peroxide generated are now relatively well understood but how the resulting low concentrations of hydrogen peroxide induce activation of multiple signaling pathways remains obscure. Several theories are presented together with accumulating evidence that 2-Cys peroxiredoxins may play a role of "effector" proteins in mediating the signaling actions of hydrogen peroxide. Identification of the mechanisms underlying these pathways offers the potential in the longer term for development of novel interventions to maintain exercise responses in the elderly with the potential to maintain muscle mass and function and consequent quality of life.

During aging loss of skeletal muscle mass and function has a si gnificant effect of an individual’s quality of life and ability to maintain independence. Both loss of muscle fibres and atrophy of the remaining fibres play a role in the mus cle decline and this is associated with loss of motor units and a reduction in the number of motor neurons. Increased oxidative damage has long been claimed to be associated with aging and many studies have reported increased amounts of oxidative damage markers are found in tissues from old organisms. Reactive oxygen species (ROS) are recognised to play a major role in cell signalling and in muscle ROS generated during contractile play an important role in signalling adaptations to contractile activity. These ’redox-regulated’ pathways are beneficial adaptations which are attenuated during aging. This review will briefly cover what is currently known about the mechanisms underlying these muscle adaptations to exercise, how they are affected by aging and assess the importan ce of these pathways in age-related loss of skeletal muscle mass and function.

The early life environment significantly affects the development of age-related skeletal muscle disorders. However, the long-term effects of lactational protein restriction on skeletal muscle are still poorly defined. Our study revealed that male mice nursed by dams fed a low-protein diet during lactation exhibited skeletal muscle growth restriction. This was associated with a dysregulation in the expression levels of genes related to the ribosome, mitochondria and skeletal muscle development. We reported that lifelong protein restriction accelerated loss of type-IIa muscle fibres and reduced muscle fibre size by impairing mitochondrial homeostasis and proteostasis at 18 months of age. However, feeding a normal-protein diet following lactational protein restriction prevented accelerated fibre loss and fibre size reduction in later life. These findings provide novel insight into the mechanisms by which lactational protein restriction hinders skeletal muscle growth and includes evidence that lifelong dietary protein restriction accelerated skeletal muscle loss in later life.

Whole-body knock-out of Cu,Zn superoxide dismutase (Sod1KO) results in accelerated, age-related loss of muscle mass and function associated with neuromuscular junction (NMJ) breakdown similar to sarcopenia. In order to determine whether altered redox in motor neurons underlies this phenotype, an inducible neuron-specific deletion of Sod1 (i-mnSod1KO) was compared with wild-type (WT) mice of different ages (adult, mid-age, and old) and whole-body Sod1KO mice. Nerve oxidative damage, motor neuron numbers and structural changes to neurons and NMJ were examined. Tamoxifen-induced deletion of neuronal Sod1 from two months of age. No specific effect of a lack of neuronal Sod1 was seen on markers of nerve oxidation (electron paramagnetic resonance of an in vivo spin probe, protein carbonyl, or protein 3-nitrotyrosine contents). i-mnSod1KO mice showed increased denervated NMJ, reduced numbers of large axons and increased number of small axons compared with old WT mice. A large proportion of the innervated NMJs in old i-mnSod1KO mice displayed a simpler structure than that seen in adult or old WT mice. Thus, previous work showed that neuronal deletion of Sod1 induced exaggerated loss of muscle in old mice, and we report that this deletion leads to a specific nerve phenotype including reduced axonal area, increased proportion of denervated NMJ, and reduced acetyl choline receptor complexity. Other changes in nerve and NMJ structure seen in the old i-mnSod1KO mice reflect aging of the mice.

Progressive muscle atrophy and loss of muscle strength associated with old age have been well documented. Although impairments in the ability of old mice to regenerate skeletal muscle following injury have been demonstrated, less is known about the way age modulates the regenerative response of motor neurons and the neuromuscular junctions (NMJ) of mice following contraction‐induced injury. The inability for NMJs to effectively regenerate following an injury could lead to the death of the denervated muscle fibers and therefore play a contributing role to age‐related sarcopenia. To investigate the relationship between age and NMJ regeneration following injury, extensor digitorum longus (EDL) muscles of adult (8‐9 months), middle‐aged (18‐19 months), and old mice (27‐28 months) were subjected to a protocol of repeated lengthening‐contractions that resulted in an acute force deficit of ~70%. After 28 days, muscles were evaluated for maximum force generating capacity, muscle mass, NMJ innervation, and endplate fragmentation. Cross sections of muscles taken at three days post injury showed extensive infiltration of mononuclear cells and widespread tissue disruption consistent with the induction of severe injury to the muscles. At 28 days, maximum isometric force recovered completely in adult mice, but was significantly reduced in middle aged and old mice when compared to muscles of adult mice. In each case, approximately 29% of fibers in the cross sections displayed containing central nuclei, whereas controls showed only 2.9%, indicating that the injured muscle fibers had undergone degeneration and regeneration. Analysis of the innervation of the NMJs in the regenerated muscles showed a moderate decrease in the number of fully innervated endplates in old mice as compared to adult and middle‐aged mice. When the architecture of the endplates in regenerated muscles was examined, the relative number of NMJs with intact postsynaptic structure varied little between the age groups, although control muscles of old mice showed an increase in the number of fragmented endplates compared with the adult and middle aged mice. Thus, the diminished ability of the skeletal muscle of old mice to recover following injury may be, in part, due to an age‐related decrease in the ability to adequately regenerate motor neurons and perhaps form NMJs in the injured muscle.

In 2021/22, the MicroAge project, a UK Space Agency-funded mission, sent 3D muscle constructs to the International Space Station to study microgravity as a model of accelerated musculoskeletal ageing on Earth. The aim of this work is to further develop the muscle constructs, compromised of human skeletal muscle cells encapsulated in fibrin hydrogels, induced to differentiate into myotubes and grown in 3D. The study uses several bioengineering approaches to determine the extent of maturity achievable in these muscle constructs in vitro, with the aim of producing a pharmacological or physiological screening platform of mature muscle, targeting muscle disorders such as sarcopenia.To enable measurement of muscle functional properties, it was also necessary to develop a suitable surrogate for force generation due to constraints of strain gauge use, and so this study also determined whether electrochemical impedance spectroscopy (EIS) was appropriate in numerous instances, opening up possibilities for rapid and automated measurement of contractile properties of in vitro skeletal muscle models. This was undertaken by the development of the MicroAge culture system to allow simultaneous measurement of force characteristic using a micro stain gauge and EIS measurements via the stimulating electrodes.The MicroAge muscle constructs have been characterised using a panel of maturation indices for skeletal muscle, including measuring contractile properties, and myosin heavy chain isoform expression using immunocytochemistry with spinning disc microscopy, RT-qPCR and Western blotting and these data will be presented.Of particular interest are the data that demonstrated a linear relationship between force generation measured using the ‘gold-standard’ approach of using a strain gauge and EIS (R 2 = 0.9), validating the use of EIS as a proxy for force generation in a range of conditions such as in muscle constructs at rest and during contraction.This allows for rapid and automated measurement of contractile properties of in vitro models of skeletal muscle, providing an exciting new platform to rapidly test pharmacological interventions that may result in improved muscle function in 3D models of disease. The study is also currently implementing bioprinting of muscle constructs to streamline construct formation and investigating varying patterns of electrical stimulation on construct maturity throughout differentiation and the results of this approach will also be presented. Supported by a PhD studentship from MRC DiMeN Doctoral Training Programme and CIMA. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.