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Electrophysiological alterations of the neuromuscular junction (NMJ) and motor unit potential (MUP) with unloading are poorly studied. We aimed to investigate these aspects and the underlying molecular mechanisms with short-term unloading and active recovery (AR). Eleven healthy males underwent a 10-day unilateral lower limb suspension (ULLS) period, followed by 21-day AR based on resistance exercise. Quadriceps femoris (QF) cross-sectional area (CSA) and isometric maximum voluntary contraction (MVC) were evaluated. Intramuscular electromyographic recordings were obtained during 10% and 25% MVC isometric contractions from the vastus lateralis (VL). Biomarkers of NMJ molecular instability (serum c-terminal agrin fragment, CAF), axonal damage (neurofilament light chain) and denervation status were assessed from blood samples and VL biopsies. NMJ and ion channel transcriptomic profiles were investigated by RNA-sequencing. QF CSA and MVC decreased with ULLS. Increased CAF and altered NMJ transcriptome with unloading suggested the emergence of NMJ molecular instability, which was not associated with impaired NMJ transmission stability. Instead, increased MUP complexity and decreased motor unit firing rates were found after ULLS. Downregulation of ion channel gene expression was found together with increased neurofilament light chain concentration and partial denervation. The AR period restored most of these neuromuscular alterations. In conclusion, the human NMJ is destabilized at the molecular level but shows functional resilience to a 10-day unloading period at least at relatively low contraction intensities. However, MUP properties are altered by ULLS, possibly due to alterations in ion channel dynamics and initial axonal damage and denervation. These changes are fully reversed by 21 days of AR. KEY POINTS: We used integrative electrophysiological and molecular approaches to comprehensively investigate changes in neuromuscular integrity and function after a 10-day unilateral lower limb suspension (ULLS), followed by 21 days of active recovery in young healthy men, with a particular focus on neuromuscular junction (NMJ) and motor unit potential (MUP) properties alterations. After 10-day ULLS, we found significant NMJ molecular alterations in the absence of NMJ transmission stability impairment. These findings suggest that the human NMJ is functionally resilient against insults and stresses induced by short-term disuse at least at relatively low contraction intensities, at which low-threshold, slow-type motor units are recruited. Intramuscular electromyography analysis revealed that unloading caused increased MUP complexity and decreased motor unit firing rates, and these alterations could be related to the observed changes in skeletal muscle ion channel pool and initial and partial signs of fibre denervation and axonal damage. The active recovery period restored these neuromuscular changes., (© 2022 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: Few days of unloading are sufficient to induce a decline of skeletal muscle mass and function; notably, contractile force is lost at a faster rate than muscle mass. The reasons behind this disproportionate loss of muscle force are still poorly understood. We provide strong evidence of two mechanisms only hypothesized until now for the rapid muscle force loss in only 10 days of bed rest. Our results show that an initial neuromuscular junction instability, accompanied by alterations in the innervation status and impairment of single fibre sarcoplasmic reticulum function contribute to the loss of contractile force in front of a preserved myofibrillar function and central activation capacity. Early onset of neuromuscular junction instability and impairment in calcium dynamics involved in excitation-contraction coupling are proposed as eligible determinants to the greater decline in muscle force than in muscle size during unloading., Abstract: Unloading induces rapid skeletal muscle atrophy and functional decline. Importantly, force is lost at a much higher rate than muscle mass. We aimed to investigate the early determinants of the disproportionate loss of force compared to that of muscle mass in response to unloading. Ten young participants underwent 10 days of bed rest (BR). At baseline (BR0) and at 10 days (BR10), quadriceps femoris (QF) volume (VOL) and isometric maximum voluntary contraction (MVC) were assessed. At BR0 and BR10 blood samples and biopsies of vastus lateralis (VL) muscle were collected. Neuromuscular junction (NMJ) stability and myofibre innervation status were assessed, together with single fibre mechanical properties and sarcoplasmic reticulum (SR) calcium handling. From BR0 to BR10, QFVOL and MVC decreased by 5.2% (P = 0.003) and 14.3% (P < 0.001), respectively. Initial and partial denervation was detected from increased neural cell adhesion molecule (NCAM)-positive myofibres at BR10 compared with BR0 (+3.4%, P = 0.016). NMJ instability was further inferred from increased C-terminal agrin fragment concentration in serum (+19.2% at BR10, P = 0.031). Fast fibre cross-sectional area (CSA) showed a trend to decrease by 15% (P = 0.055) at BR10, while single fibre maximal tension (force/CSA) was unchanged. However, at BR10 SR Ca 2+ release in response to caffeine decreased by 35.1% (P < 0.002) and 30.2% (P < 0.001) in fast and slow fibres, respectively, pointing to an impaired excitation-contraction coupling. These findings support the view that the early onset of NMJ instability and impairment in SR function are eligible mechanisms contributing to the greater decline in muscle force than in muscle size during unloading., (© 2021 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Neural conditioning to scenarios of muscle disuse is undoubtedly a cause of functional decrements that typically exceed losses of muscle size. Yet establishing the relative contribution of neural adaptation and the specific location in the motor pathway remains technically challenging. Several studies of healthy humans have targeted this system and have established that motor unit firing rate is suppressed following disuse, with a number of critical caveats. It is suppressed in the immobilized limb only, at relative and absolute force levels, and preferentially targets lower-threshold motor units. Concomitantly, electrophysiological investigation of neuromuscular junction transmission (NMJ) stability of lower-threshold motor units reveals minimal change following disuse. These findings contrast with numerous other methods, which show clear involvement of the NMJ but are unable to characterize the motor unit to which they belong. It is physiologically plausible that decrements observed following disuse are a result of suppressed firing rate of lower-threshold motor units and impairment of transmission of the NMJ of higher-threshold motor units. As such, motor units within the pool should be viewed in light of their varying susceptibility to disuse., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

The removal of skeletal muscle tension (unloading or disuse) is followed by many changes in the neuromuscular system, including muscle atrophy and loss of isometric maximal strength (measured by maximal force, F max ). Explosive strength, i.e. the ability to develop the highest force in the shortest possible time, to maximise rate of force development (RFD), is a fundamental neuromuscular capability, often more functionally relevant than maximal muscle strength. In the present review, we discuss data from studies that looked at the effect of muscle unloading on isometric maximal versus explosive strength. We present evidence that muscle unloading yields a greater decline in explosive relative to maximal strength. The longer the unloading duration, the smaller the difference between the decline in the two measures. Potential mechanisms that may explain the greater decline in measures of RFD relative to F max after unloading are higher recruitment thresholds and lower firing rates of motor units, slower twitch kinetics, impaired excitation-contraction coupling, and decreased tendon stiffness. Using a Hill-type force model, we showed that this ensemble of adaptations minimises the loss of force production at submaximal contraction intensities, at the expense of a disproportionately lower RFD. With regard to the high functional relevance of RFD on one hand, and the boosted detrimental effects of inactivity on RFD on the other hand, it seems crucial to implement specific exercises targeting explosive strength in populations that experience muscle disuse over a longer time., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

A growing body of research has provided evidence that de novo expression of connexin hemichannels and upregulation of pannexin hemichannels (Cx HCs and Panx HCs, respectively) in the cytoplasmic membrane of skeletal muscle (sarcolemma) are critical steps in the pathogenesis of muscle dysfunction of many genetic and acquired muscle diseases. This review provides an overview of the current understanding of the molecular mechanisms regulating the expression of Cx and Panx HCs in skeletal muscle, as well as their roles in both muscle physiology and pathologies. Additionally, it addresses existing gaps in knowledge and outlines future challenges in the field., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

Skeletal muscle wasting is the hallmark pathophysiological adaptation to unloading or disuse that demonstrates the dependency on frequent mechanical stimulation (e.g. muscle activation and subsequent loading) for homeostasis of normally load-bearing muscles. In the absence of mitigation strategies, no mammalian organism is resistant to muscle atrophy driven by unloading. Given the profound impact of unloading-induced muscle wasting on physical capacity, metabolic health and immune function; mitigation strategies during unloading and/or augmentation approaches during recovery have broad healthcare implications in settings of bed-bound hospitalization, cast immobilization and spaceflight. This topical review aims to: (1) provide a succinct, state-of-the-field summary of seminal and recent findings regarding the mechanisms of unloading-induced skeletal muscle wasting; (2) discuss unsuccessful vs. promising mitigation and recovery augmentation strategies; and (3) identify knowledge gaps ripe for future research. We focus on the rapid muscle atrophy driven by relatively short-term mechanical unloading/disuse, which is in many ways mechanistically distinct from both hypermetabolic muscle wasting and denervation-induced muscle atrophy. By restricting this discussion to mechanical unloading during which all components of the nervous system remain intact (e.g. without denervation models), mechanical loading requiring motor and sensory neural circuits in muscle remain viable targets for both mitigation and recovery augmentation. We emphasize findings in humans with comparative discussions of studies in rodents which enable elaboration of key mechanisms. We also discuss what is currently known about the effects of age and sex as biological factors, and both are highlighted as knowledge gaps and novel future directions due to limited research., (© 2024 The Authors. The Journal of Physiology © 2024 The Physiological Society.)

Mitochondrial dysfunctions are thought to contribute to muscle atrophy and weakness that develop during ageing and mechanical unloading caused by immobilization, bed rest and microgravity. Older adults are at greater risk of developing muscle and mitochondrial dysfunctions in response to unloading. Although exercise is well known to promote muscle and mitochondrial health, its protective effect during mechanical unloading in older adults remains largely unexplored. Here, we investigated the impact of 14 days of head-down tilt bed rest (HDBR) with and without a multimodal exercise countermeasure in older men and women (55-65 years). Leg muscle volume was assessed using magnetic resonance imaging. Biopsies of the vastus lateralis were performed to assess markers of mitochondrial content, respiration, reactive oxygen species (ROS) production and calcium retention capacity (mCRC). Indices of mitochondrial quality control (MQC), including markers of fusion (MFN1 and 2), fission (Drp1), mitophagy (Parkin) and autophagy (p62 and LC3I and II) were measured using immunoblots. Muscle cross-sections were stained for neural cell adhesion molecule (NCAM, a marker of denervation). HDBR triggered muscle atrophy, decreased mitochondrial content and respiration and increased mitochondrial ROS production. HDBR had no impact on mCRC or MQC markers but increased markers of autophagy and denervation. Exercise prevented the deleterious effects of HDBR on leg muscle volume, mitochondrial ROS production and markers of autophagy and denervation. Exercise also increased mitochondrial content and respiration without altering mCRC and MQC markers. Collectively, our results indicate that an exercise countermeasure that can be performed in bed is effective in protecting muscle and mitochondrial health during HDBR in older adults. KEY POINTS: Conditions associated with muscle unloading, such as immobilization, bed rest or microgravity, result in muscle atrophy and weakness, particularly in older adults. Mitochondrial dysfunctions are thought to contribute to muscle atrophy caused by unloading and ageing. However, whether exercise can counteract the deleterious effects of unloading in older adults remains largely unexplored. Here, we report that older adults exposed to 14 days of head-down tilt bed rest (HDBR) displayed upper leg muscle atrophy, a decrease in mitochondrial content and respiration, an increase in H 2 O 2 emission, and an increase in autophagy and denervation markers. No impact of HDBR on mitochondrial quality control was observed. A multimodal exercise countermeasure prevented the deleterious effects of HDBR on upper leg muscle volume, mitochondrial reactive oxygen species emission, and markers of autophagy and denervation and increased mitochondrial content and respiration. These findings highlight the effectiveness of exercise in promoting muscle and mitochondrial health in older adults undergoing bed rest., (© 2024 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Disuse atrophy, caused by situations of unloading such as limb immobilisation, causes a rapid yet diverging reduction in skeletal muscle function when compared to muscle mass. While mechanistic insight into the loss of mass is well studied, deterioration of muscle function with a focus towards the neural input to muscle remains underexplored. This study aimed to determine the role of motor unit adaptation in disuse‐induced neuromuscular deficits. Ten young, healthy male volunteers underwent 15 days of unilateral lower limb immobilisation with intramuscular electromyography (iEMG) bilaterally recorded from the vastus lateralis (VL) during knee extensor contractions normalised to maximal voluntary contraction (MVC), pre and post disuse. Muscle cross‐sectional area was determined by ultrasound. Individual MUs were sampled and analysed for changes in motor unit (MU) discharge and MU potential (MUP) characteristics. VL CSA was reduced by approximately 15% which was exceeded by a two‐fold decrease of 31% in muscle strength in the immobilised limb, with no change in either parameter in the non‐immobilised limb. Parameters of MUP size were reduced by 11% to 24% with immobilisation, while neuromuscular junction (NMJ) transmission instability remained unchanged, and MU firing rate decreased by 8% to 11% at several contraction levels. All adaptations were observed in the immobilised limb only. These findings highlight impaired neural input following immobilisation reflected by suppressed MU firing rate which may underpin the disproportionate reductions of strength relative to muscle size. Key points: Muscle mass and function decline rapidly in situations of disuse such as bed rest and limb immobilisation.The reduction in muscle function commonly exceeds that of muscle mass, which may be associated with the dysregulation of neural input to muscle.We have used intramuscular electromyography to sample individual motor unit and near fibre potentials from the vastus lateralis following 15 days of unilateral limb immobilisation. Following disuse, the disproportionate loss of muscle strength when compared to size coincided with suppressed motor unit firing rate.These motor unit adaptations were observed at multiple contraction levels and in the immobilised limb only. Our findings demonstrate neural dysregulation as a key component of functional loss following muscle disuse in humans. [ABSTRACT FROM AUTHOR]

A motor unit (MU) comprises the neuron cell body, its corresponding axon and each of the muscle fibres it innervates. Many studies highlight age‐related reductions in the number of MUs, yet the ability of a MU to undergo remodelling and to expand to rescue denervated muscle fibres is also a defining feature of MU plasticity. Remodelling of MUs involves two coordinated processes: (i) axonal sprouting and new branching growth from adjacent surviving neurons, and (ii) the formation of key structures around the neuromuscular junction to resume muscle–nerve communication. These processes rely on neurotrophins and coordinated signalling in muscle–nerve interactions. To date, several neurotrophins have attracted focus in animal models, including brain‐derived neurotrophic factor and insulin‐like growth factors I and II. Exercise in older age has demonstrated benefits in multiple physiological systems including skeletal muscle, yet evidence suggests this may also extend to peripheral MU remodelling. There is, however, a lack of research in humans due to methodological limitations which are easily surmountable in animal models. To improve mechanistic insight of the effects of exercise on MU remodelling with advancing age, future research should focus on combining methodological approaches to explore the in vivo physiological function of the MU alongside alterations of the localised molecular environment. [ABSTRACT FROM AUTHOR]

Muscle fibre denervation and declining numbers of muscle stem (satellite) cells are defining characteristics of ageing skeletal muscle. The aim of this study was to investigate the potential for lifelong recreational exercise to offset muscle fibre denervation and compromised satellite cell content and function, both at rest and under challenged conditions. Sixteen elderly lifelong recreational exercisers (LLEX) were studied alongside groups of age‐matched sedentary (SED) and young subjects. Lean body mass and maximal voluntary contraction were assessed, and a strength training bout was performed. From muscle biopsies, tissue and primary myogenic cell cultures were analysed by immunofluorescence and RT‐qPCR to assess myofibre denervation and satellite cell quantity and function. LLEX demonstrated superior muscle function under challenged conditions. When compared with SED, the muscle of LLEX was found to contain a greater content of satellite cells associated with type II myofibres specifically, along with higher mRNA levels of the beta and gamma acetylcholine receptors (AChR). No difference was observed between LLEX and SED for the proportion of denervated fibres or satellite cell function, as assessed in vitro by myogenic cell differentiation and fusion index assays. When compared with inactive counterparts, the skeletal muscle of lifelong exercisers is characterised by greater fatigue resistance under challenged conditions in vivo, together with a more youthful tissue satellite cell and AChR profile. Our data suggest a little recreational level exercise goes a long way in protecting against the emergence of classic phenotypic traits associated with the aged muscle. Key points: The detrimental effects of ageing can be partially offset by lifelong self‐organized recreational exercise, as evidence by preserved type II myofibre‐associated satellite cells, a beneficial muscle innervation status and greater fatigue resistance under challenged conditions.Satellite cell function (in vitro), muscle fibre size and muscle fibre denervation determined by immunofluorescence were not affected by recreational exercise.Individuals that are recreationally active are far more abundant than master athletes, which sharply increases the translational perspective of the present study. Future studies should further investigate recreational activity in relation to muscle health, while also including female participants. [ABSTRACT FROM AUTHOR]

In order to identify peripheral biomarkers of impaired oxidative metabolism during exercise following a 10‐day bed rest, 10 males performed an incremental exercise (to determine peak pulmonary V̇O2 (V̇O2p)) and moderate‐intensity exercises, before (PRE) and after (POST) bed rest. Blood flow response was evaluated in the common femoral artery by Eco‐Doppler during 1 min of passive leg movements (PLM). The intramuscular matching between O2 delivery and O2 utilization was evaluated by near‐infrared spectroscopy (NIRS). Mitochondrial respiration was evaluated ex vivo by high‐resolution respirometry in isolated muscle fibres, and in vivo by NIRS by the evaluation of skeletal muscle V̇O2 (V̇O2m) recovery kinetics. Resting V̇O2m was estimated by NIRS. Peak V̇O2p was lower in POST vs. PRE. The area under the blood flow vs. time curve during PLM was smaller (P = 0.03) in POST (274 ± 233 mL) vs. PRE (427 ± 291). An increased (P = 0.03) overshoot of muscle deoxygenation during a metabolic transition was identified in POST. Skeletal muscle citrate synthase activity was not different (P = 0.11) in POST (131 ± 16 nmol min–1 mg–1) vs. PRE (138 ± 19). Maximal ADP‐stimulated mitochondrial respiration (66 ± 18 pmol s–1 mg–1 (POST) vs. 72 ± 14 (PRE), P = 0.41) was not affected by bed rest. Apparent Km for ADP sensitivity of mitochondrial respiration was reduced in POST vs. PRE (P = 0.04). The V̇O2m recovery time constant was not different (P = 0.79) in POST (22 ± 6 s) vs. PRE (22 ± 6). Resting V̇O2m was reduced by 25% in POST vs. PRE (P = 0.006). Microvascular‐endothelial function was impaired following a 10‐day bed rest, whereas mitochondrial mass and function (both in vivo and ex vivo) were unaffected or slightly enhanced. Key points: Ten days of horizontal bed rest impaired in vivo oxidative function during exercise.Microvascular impairments were identified by different methods.Mitochondrial mass and mitochondrial function (evaluated both in vivo and ex vivo) were unchanged or even improved (i.e. enhanced mitochondrial sensitivity to submaximal [ADP]).Resting muscle oxygen uptake was significantly lower following bed rest, suggesting that muscle catabolic processes induced by bed rest/inactivity are less energy‐consuming than anabolic ones. [ABSTRACT FROM AUTHOR]

Atrophy, bedrest, calcium, disuse, neuromuscular junction, sarcopenia, skeletal muscle, unloading Keywords: atrophy; bedrest; calcium; disuse; neuromuscular junction; sarcopenia; skeletal muscle; unloading EN atrophy bedrest calcium disuse neuromuscular junction sarcopenia skeletal muscle unloading 4249 4251 3 09/16/21 20210915 NES 210915 Maintaining skeletal muscle mass is essential for normal ambulatory function and metabolism. These data, together with altered expression of genes related to NMJ (e.g. AGRN, CHRNA1 and HOMER2), suggest that just 10 days of bedrest may be sufficient to result in a loss of NMJ stability. [Extracted from the article]

Keywords: denervation; disuse; muscle function; neuromuscular EN denervation disuse muscle function neuromuscular 4539 4540 2 11/03/22 20221101 NES 221101 Most people will, throughout their lifetime, experience periods of muscle unloading, disuse and inactivity, relating to disease, injury or hospitalization. Next, the authors analysed blood samples and vastus lateralis muscle biopsies for markers of impaired synaptic transmission. Inns et al. (2022) recruited 10 men (mean ± SD, 23.7 ± 3.4 years of age), who underwent 15 days of unilateral limb immobilization using a leg brace on one leg (experimental leg), and an air-boot on the other leg (control leg). [Extracted from the article]