Effects of short-term unloading and active recovery on human motor unit properties, neuromuscular junction transmission and transcriptomic profile.

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.

Neuromuscular junction instability and altered intracellular calcium handling as early determinants of force loss during unloading in humans.

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 Ca2+ 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.

Peripheral nerve adaptations to 10 days of horizontal bed rest in healthy young adult males.

Space analogs, such as bed rest, are used to reproduce microgravity-induced morphological and physiological changes and can be used as clinical models of prolonged inactivity. Nevertheless, nonuniform decreases in muscle mass and function have been frequently reported, and peripheral nerve adaptations have been poorly studied, although some of these mechanisms may be explained. Ten young healthy males (18-33 yr) underwent 10 days of horizontal bed rest. Peripheral neurophysiological assessments were performed bilaterally for the dominant (DL) and nondominant upper and lower limbs (N-DL) on the 1st and 10th day of bed rest, including ultrasound of the median, deep peroneal nerve (DPN), and common fibular nerve (CFN) , as well as a complete nerve conduction study (NCS) of the upper and lower limbs. Consistently, reduced F waves, suggesting peripheral nerve dysfunction, of both the peroneal (DL: P = 0.005, N-DL: P = 0.013) and tibial nerves (DL: P = 0.037, N-DL: P = 0.005) were found bilaterally, whereas no changes were observed in nerve ultrasound or other parameters of the NCS of both the upper and lower limbs. In these young healthy males, only the F waves, known to respond to postural changes, were significantly affected by short-term bed rest. These preliminary results suggest that during simulated microgravity, most changes occur at the muscle or central nervous system level. Since the assessment of F waves is common in clinical neurophysiological examinations, caution should be used when testing individuals after prolonged immobility.

Longitudinal hypertrophic and transcriptional responses to high-load eccentric-concentric vs concentric training in males.

High-load eccentric training reputedly produces greater muscle hypertrophy than concentric training, possibly due to greater loading and/or inflammation. We quantified the temporal impact of combined maximal concentric-eccentric training vs maximal concentric training on muscle cross-sectional area (CSA), volume, and targeted mRNA expression (93 transcripts). Eight recreationally active males (24 ± 5 years, BMI 23.5 ± 2.5 kg/m2 ) performed 3 x 30 maximal eccentric isokinetic knee extensions and 2 x 30 maximal concentric knee extensions in dominant limb (ECC + CON) and 5 x 30 maximal concentric contractions (CON) in the non-dominant limb for 12 weeks (all 90°/s, 3x/wk). Quadriceps muscle CSA and volume were measured at baseline, 28 days (d), and 84 d in both limbs (3T MRI). Resting vastus lateralis biopsies were obtained from both limbs at baseline, 24 hours (h), 7, 28, and 84 d for mRNA abundance measurements (RT-PCR microfluidic cards). Work output was greater throughout training in ECC + CON vs CON (20.8 ± 9.7%, P < .001). Muscle CSA increased from baseline in both limbs at 28 d (CON 4.3 ± 2.6%, ECC + CON 4.0 ± 1.9%, both P < .001) and 84d (CON 3.9 ± 2.3%, ECC + CON 4.0 ± 3.1%, both P < .001), and muscle volume and isometric strength at 84 d (CON 44.8 ± 40.0%, P < .001; ECC + CON 36.9 ± 40.0%, P < .01), but no between-limb differences existed in any parameter. Ingenuity Pathway Analysis identified several cellular functions associated with regulation of muscle mass and metabolism as altered by both modalities at 24 h and 7 d, but particularly with ECC + CON. However, mRNA responses waned thereafter, regardless of modality. Initial muscle mRNA responses to training did not reflect chronic training-induced hypertrophy. Moreover, ECC + CON did not produce greater hypertrophy than CON, despite greater loading throughout and a differential mRNA response during the initial training week.

Remodeling the Skeletal Muscle Extracellular Matrix in Older Age-Effects of Acute Exercise Stimuli on Gene Expression.

With advancing age, the skeletal muscle extracellular matrix (ECM) undergoes fibrotic changes that may lead to increased muscle stiffness, injury susceptibility and strength loss. This study tested the potential of different exercises to counter these changes by stimulating the activity of genes associated with ECM remodeling. Twenty-six healthy men (66.9 ± 3.9 years) were stratified to two of four groups, performing unilateral (i) conventional resistance exercise, (ii) conventional resistance exercise followed by self-myofascial release (CEBR), (iii) eccentric-only exercise (ECC) or (iv) plyometric jumps (PLY). The non-trained leg served as control. Six hours post-exercise, vastus lateralis muscle biopsy samples were analyzed for the expression of genes associated with ECM collagen synthesis (COL1A1), matrix metallopeptidases (collagen degradation; MMPs) and peptidase inhibitors (TIMP1). Significant between-group differences were found for MMP3, MMP15 and TIMP1, with the greatest responses in MMP3 and TIMP1 seen in CEBR and in MMP15 in ECC. MMP9 (3.24-3.81-fold change) and COL1A1 (1.47-2.40-fold change) were increased in CEBR and PLY, although between-group differences were non-significant. The expression of ECM-related genes is exercise-specific, with CEBR and PLY triggering either earlier or stronger remodeling than other stimuli. Training studies will test whether execution of such exercises may help counter age-associated muscle fibrosis.

Assessing sarcopenia with vastus lateralis muscle ultrasound: an operative protocol.

BACKGROUND: Muscle ultrasound (MUS) has so far not been implemented for sarcopenia assessment in clinical geriatric practice due to allegedly low reproducibility of results in the absence of standardization of procedures. However, rigorous and standardized application of this technique yields highly reproducible results. Its application, especially if integrated with clinical evaluation and comprehensive geriatric assessment, proofs very useful for rapidly obtaining information on muscle mass and architecture. OBJECTIVE: Here, we present a standardized protocol for performing right vastus lateralis (RVL) MUS and measuring parameters of muscle size and architecture. METHODS: RVL muscle thickness (MT), fascicle length (FL), pennation angle (PA), echo-intensity (EI) and cross-sectional area (CSA) can be assessed with this protocol. A portable instrument equipped with a 5-cm long 3-11 mHz linear probe should be used with both B-mode real-time and extended-field-of-view (EFOV) techniques. Longitudinal B-mode and transverse EFOV images should be acquired during each exam, and analyzed with NIH-ImageJ software. CONCLUSIONS: This operative protocol represents a good compromise between the feasibility of MUS in clinical settings and the need of obtaining precise measurements of muscle parameters. Future studies should verify the reproducibility of the proposed technique, and its correlation with appendicular lean mass and parameters of muscle function.

Differential expression of perilipin 2 and 5 in human skeletal muscle during aging and their association with atrophy-related genes.

Sarcopenia, the progressive loss of muscle mass and strength, is a phenomenon characterizing human aging whose etiology is still not clear. While there is increasing evidence for the influence of inter-muscular adipose tissue infiltration in the development of sarcopenia, much less is known about a possible role for intra-muscular triglycerides (IMTG). IMTG accumulate in form of lipid droplets decorated by proteins such as Perilipins (Plins). In skeletal muscle the most abundant are Plin2 and Plin5. In this study we compared the expression of these two Plins in Vastus lateralis muscle samples of subjects of different age, both healthy donors (HD) and patients with limited lower limb mobility (LLMI). These latter are characterized by a condition of chronic physical inactivity. Plin2 expression resulted higher in old age for both HD and LLMI patients, while Plin5 slightly decreased only in LLMI patients. Moreover, in these patients, only Plin2 was associated with the decrease of muscle strength and the expression of factors related to muscle atrophy (MuRF1, Atrogin and p53). An increase in Plin2 and a concomitant decrease of Plin5 was also observed when we considered animal model of disuse-induced muscle atrophy. As a whole, these data indicate that Plin2 and Plin5 have a different expression pattern during muscle aging and inactivity, and only Plin2 appears to be associated with functional alterations of the muscle.

The Impact of Different Diagnostic Criteria on the Prevalence of Sarcopenia in Healthy Elderly Participants and Geriatric Outpatients.

BACKGROUND: A consensus on the diagnostic criteria for sarcopenia, a common syndrome in the elderly, has not been reached yet. Prevalence rates vary between studies due to the use of different criteria encompassing different measures, correction factors and cutoff points. OBJECTIVE: This study compared prevalence rates of sarcopenia using nine sets of diagnostic criteria applied in two different elderly populations. METHODS: The study population encompassed 308 healthy elderly participants (152 males, 156 females; mean age 74 years) and 123 geriatric outpatients (54 males, 69 females; mean age 81 years). Diagnostic criteria included relative muscle mass, absolute muscle mass, muscle strength and physical performance. RESULTS: Prevalence rates of sarcopenia varied between 0 and 15% in healthy elderly participants and between 2 and 34% in geriatric outpatients. CONCLUSION: This study clearly demonstrates the dependency of sarcopenia prevalence rates on the applied diagnostic criteria.

A validation of the application of D(2)O stable isotope tracer techniques for monitoring day-to-day changes in muscle protein subfraction synthesis in humans.

Quantification of muscle protein synthesis (MPS) remains a cornerstone for understanding the control of muscle mass. Traditional [(13)C]amino acid tracer methodologies necessitate sustained bed rest and intravenous cannulation(s), restricting studies to ~12 h, and thus cannot holistically inform on diurnal MPS. This limits insight into the regulation of habitual muscle metabolism in health, aging, and disease while querying the utility of tracer techniques to predict the long-term efficacy of anabolic/anticatabolic interventions. We tested the efficacy of the D2O tracer for quantifying MPS over a period not feasible with (13)C tracers and too short to quantify changes in mass. Eight men (22 ± 3.5 yr) undertook one-legged resistance exercise over an 8-day period (4 × 8-10 repetitions, 80% 1RM every 2nd day, to yield "nonexercised" vs. "exercise" leg comparisons), with vastus lateralis biopsies taken bilaterally at 0, 2, 4, and 8 days. After day 0 biopsies, participants consumed a D2O bolus (150 ml, 70 atom%); saliva was collected daily. Fractional synthetic rates (FSRs) of myofibrillar (MyoPS), sarcoplasmic (SPS), and collagen (CPS) protein fractions were measured by GC-pyrolysis-IRMS and TC/EA-IRMS. Body water initially enriched at 0.16-0.24 APE decayed at ~0.009%/day. In the nonexercised leg, MyoPS was 1.45 ± 0.10, 1.47 ± 0.06, and 1.35 ± 0.07%/day at 0-2, 0-4, and 0-8 days, respectively (~0.05-0.06%/h). MyoPS was greater in the exercised leg (0-2 days: 1.97 ± 0.13%/day; 0-4 days: 1.96 ± 0.15%/day, P < 0.01; 0-8 days: 1.79 ± 0.12%/day, P < 0.05). CPS was slower than MyoPS but followed a similar pattern, with the exercised leg tending to yield greater FSRs (0-2 days: 1.14 ± 0.13 vs. 1.45 ± 0.15%/day; 0-4 days: 1.13 ± 0.07%/day vs. 1.47 ± 0.18%/day; 0-8 days: 1.03 ± 0.09%/day vs. 1.40 ± 0.11%/day). SPS remained unchanged. Therefore, D2O has unrivaled utility to quantify day-to-day MPS in humans and inform on short-term changes in anabolism and presumably catabolism alike.

Knee extensor fatigue resistance of young and older men and women performing sustained and brief intermittent isometric contractions.

INTRODUCTION: Susceptibility to muscle fatigue during aging could depend on muscle activation patterns. METHODS: Young (mean age, 22 years) and older (mean age 70 years) men and women completed two fatigue tests of knee extensor muscles using voluntary and electrically stimulated contractions. RESULTS: Older subjects displayed a shift to the left of the torque-frequency relationship and held a sustained voluntary isometric contraction at 50% maximal strength for significantly longer than young (P < 0.001). Young and old showed similar fatigue during electrically induced, intermittent isometric contractions (1-s on, 1-s off for 2 min), but women fatigued less than men (P = 0.001). Stronger muscles fatigued more quickly, and slower contractile properties were associated with longer sustained contractions. CONCLUSIONS: The slowing and weakness of older muscle was associated with superior fatigue resistance during sustained isometric contractions. Young and old showed similar fatigue following a series of brief, intermittent contractions, but women fatigued less than men.

Factors affecting metabolic cost of transport during a multi-stage running race.

The aim of this study was to investigate: (1) the role of , the fraction of (F) and the metabolic cost of transport (CoT) in determining performance during an ultra-endurance competition and (2) the effects of the race on several biomechanical and morphological parameters of the lower limbs that are likely to affect CoT. Eleven runners (aged 29-54 years) participated in an ultra-endurance competition consisting of three running stages of 25, 55 and 13 km on three consecutive days. Anthropometric characteristics, body composition, morphological properties of the gastrocnemius medialis, maximal explosive power of the lower limb and were determined before the competition. In addition, biomechanics of running and CoT were determined, before and immediately after each running stage. Performance was directly proportional to (r=0.77) and F (r=0.36), and inversely proportional to CoT (r=-0.30). Low CoT values were significantly related to high maximal power of the lower limbs (r=-0.74) and vertical stiffness (r=-0.65) and low footprint index (FPI, r=0.70), step frequency (r=0.62) and external work (r=0.60). About 50% of the increase in CoT during the stages of the competition was accounted for by changes in FPI, which represents a global evaluation of medio-lateral displacement of the foot during the whole stance phase, which in turn is associated with the myotendinous characteristics of the lower limb. Thus, lower CoT values were related to greater muscular power and lower FPI, suggesting that a better ankle stability is likely to achieve better performance in an ultra-endurance running competition.

Neuromodulatory Contribution to Muscle Force Production after Short-Term Unloading and Active Recovery.

PURPOSE: Prior evidence has shown that neural factors contribute to the loss of muscle force after skeletal muscle disuse. However, little is known about the specific neural mechanisms altered by disuse. Persistent inward current (PIC) is an intrinsic property of motoneurons responsible for prolonging and amplifying the synaptic input, proportionally to the level of neuromodulation, thus influencing motoneuron discharge rate and force production. Here, we hypothesized that short-term unilateral lower limb suspension (ULLS) would reduce the neuromodulatory input associated with PIC, contributing to the reduction of force generation capacity. In addition, we tested whether physical exercise would restore the force generation capacity by reestablishing the initial level of neuromodulatory input. METHODS: In 12 young adults, we assessed maximal voluntary contraction pre- and post-10 d of ULLS and after 21 d of active recovery (AR) based on resistance exercise. PIC was estimated from high-density surface electromyograms of the vastus lateralis muscle as the delta frequency (Δ F ) of paired motor units calculated during isometric ramped contractions. RESULTS: The values of Δ F were reduced after 10 d of ULLS (-33%, P < 0.001), but were fully reestablished after the AR (+29.4%, P < 0.001). The changes in estimated PIC values were correlated ( r = 0.63, P = 0.004) with the reduction in maximal voluntary contraction after ULLS (-29%, P = 0.002) and its recovery after the AR (+28.5%, P = 0.003). CONCLUSIONS: Our findings suggest that PIC estimates are reduced by muscle disuse and may contribute to the loss of force production and its recovery with exercise. Overall, this is the first study demonstrating that, in addition to peripheral neuromuscular changes, central neuromodulation is a major contributor to the loss of force generation capacity after disuse, and can be recovered after resistance exercise.

Spaceflight on the ISS changed the skeletal muscle proteome of two astronauts.

Skeletal muscle undergoes atrophy and loss of force during long space missions, when astronauts are persistently exposed to altered gravity and increased ionizing radiation. We previously carried out mass spectrometry-based proteomics from skeletal muscle biopsies of two astronauts, taken before and after a mission on the International Space Station. The experiments were part of an effort to find similarities between spaceflight and bed rest, a ground-based model of unloading, focused on proteins located at the costameres. We here extend the data analysis of the astronaut dataset and show compartment-resolved changes in the mitochondrial proteome, remodeling of the extracellular matrix and of the antioxidant response. The astronauts differed in their level of onboard physical exercise, which correlated with their respective preservation of muscle mass and force at landing in previous analyses. We show that the mitochondrial proteome downregulation during spaceflight, particularly the inner membrane and matrix, was dramatic for both astronauts. The expression of autophagy regulators and reactive oxygen species scavengers, however, showed partially opposite expression trends in the two subjects, possibly correlating with their level of onboard exercise. As mitochondria are primarily affected in many different tissues during spaceflight, we hypothesize that reactive oxygen species (ROS) rather than mechanical unloading per se could be the primary cause of skeletal muscle mitochondrial damage in space. Onboard physical exercise might have a strong direct effect on the prevention of muscle atrophy through mechanotransduction and a subsidiary effect on mitochondrial quality control, possibly through upregulation of autophagy and anti-oxidant responses.

Lower limb suspension induces threshold-specific alterations of motor units properties that are reversed by active recovery.

PURPOSE: This study aimed to non-invasively test the hypothesis that (a) short-term lower limb unloading would induce changes in the neural control of force production (based on motor units (MUs) properties) in the vastus lateralis muscle and (b) possible changes are reversed by active recovery (AR). METHODS: Ten young males underwent 10 days of unilateral lower limb suspension (ULLS) followed by 21 days of AR. During ULLS, participants walked exclusively on crutches with the dominant leg suspended in a slightly flexed position (15°-20°) and with the contralateral foot raised by an elevated shoe. The AR was based on resistance exercise (leg press and leg extension) and executed at 70% of each participant's 1 repetition maximum, 3 times/week. Maximal voluntary isometric contraction (MVC) of knee extensors and MUs properties of the vastus lateralis muscle were measured at baseline, after ULLS, and after AR. MUs were identified using high-density electromyography during trapezoidal isometric contractions at 10%, 25%, and 50% of the current MVC, and individual MUs were tracked across the 3 data collection points. RESULTS: We identified 1428 unique MUs, and 270 of them (18.9%) were accurately tracked. After ULLS, MVC decreased by 29.77%, MUs absolute recruitment/derecruitment thresholds were reduced at all contraction intensities (with changes between the 2 variables strongly correlated), while discharge rate was reduced at 10% and 25% but not at 50% MVC. Impaired MVC and MUs properties fully recovered to baseline levels after AR. Similar changes were observed in the pool of total as well as tracked MUs. CONCLUSION: Our novel results demonstrate, non-invasively, that 10 days of ULLS affected neural control predominantly by altering the discharge rate of lower-threshold but not of higher-threshold MUs, suggesting a preferential impact of disuse on motoneurons with a lower depolarization threshold. However, after 21 days of AR, the impaired MUs properties were fully restored to baseline levels, highlighting the plasticity of the components involved in neural control.

Costamere remodeling with muscle loading and unloading in healthy young men.

Costameres are mechano-sensory sites of focal adhesion in the sarcolemma that provide a structural anchor for myofibrils. Their turnover is regulated by integrin-associated focal adhesion kinase (FAK). We hypothesized that changes in content of costamere components (beta 1 integrin, FAK, meta-vinculin, gamma-vinculin) with increased and reduced loading of human anti-gravity muscle would: (i) relate to changes in muscle size and molecular parameters of muscle size regulation [p70S6K, myosin heavy chain (MHC)1 and MHCIIA]; (ii) correspond to adjustments in activity and expression of FAK, and its negative regulator, FRNK; and (iii) reflect the temporal response to reduced and increased loading. Unloading induced a progressive decline in thickness of human vastus lateralis muscle after 8 and 34 days of bedrest (-4% and -14%, respectively; n = 9), contrasting the increase in muscle thickness after 10 and 27 days of resistance training (+5% and +13%; n = 6). Changes in muscle thickness were correlated with changes in cross-sectional area of type I muscle fibers (r = 0.66) and beta 1 integrin content (r = 0.76) at the mid-point of altered loading. Changes in meta-vinculin and FAK-pY397 content were correlated (r = 0.85) and differed, together with the changes of beta 1 integrin, MHCI, MHCII and p70S6K, between the mid- and end-point of resistance training. By contrast, costamere protein level changes did not differ between time points of bedrest. The findings emphasize the role of FAK-regulated costamere turnover in the load-dependent addition and removal of myofibrils, and argue for two phases of muscle remodeling with resistance training, which do not manifest at the macroscopic level.

Physiological and functional evaluation of healthy young and older men and women: design of the European MyoAge study.

Within the European multi-centre MyoAge project, one workpackage was designed to investigate the contribution of age-related changes to muscle mass, contractile characteristics and neural control in relation to reductions in mobility in older age. The methodology has been described here. Test centres were located in Manchester, UK; Paris, France; Leiden, The Netherlands; Tartu, Estonia and Jyväskylä, Finland. In total, 182 young (18-30 years old, 52.2 % female) and 322 older adults (69-81 years old, 50 % female) have been examined. The participants were independent living, socially active and free from disease that impaired mobility levels. The older participants were selected based on physical activity levels, such that half exceeded current recommended physical activity levels and the other half had lower physical activity levels than is recommended to maintain health. Measurements consisted of blood pressure; anthropometry and body composition (dual-energy X-ray absorptiometry and magnetic resonance imaging); lung function; standing balance and cognitive function (CANTAB). Mobility was assessed using the Timed Up and Go, a 6 min walk, activity questionnaires and accelerometers to monitor habitual daily activities. Muscle strength, power, fatigue and neural activation were assessed using a combination of voluntary and electrically stimulated contractions. Fasting blood samples and skeletal muscle biopsies were collected for detailed examination of cell and molecular differences between young and older individuals. The results from this study will provide a detailed insight into "normal, healthy" ageing, linking whole-body function to the structure and function of the neuromuscular system and the molecular characteristics of skeletal muscle.

Circulating levels of adipokines and IGF-1 are associated with skeletal muscle strength of young and old healthy subjects.

It is known that adipose tissue mass increases with age, and that a number of hormones, collectively called adipokines, are produced by adipose tissue. For most of them it is not known whether their plasmatic levels change with age. Moreover, it is known that adipose tissue infiltration in skeletal muscle is related to sarcopenia and loss of muscle strength. In this study we investigated the age-related changes of representative adipokines and insulin-like growth factor (IGF)-1 and their effect on muscle strength. We studied the association between circulating levels of adiponectin, leptin, resistin and IGF-1 and muscle strength. This cross-sectional study included 412 subjects of different age (152 subjects aged 18-30 years and 260 subjects aged 69-81 years) recruited within the framework of the European research network project "Myoage". The levels of adiponectin (both in male and female subjects) and leptin (only in males) were significantly higher in old subjects compared to young, while those of IGF-1 were lower in old subjects. In old subjects adiponectin, resistin and the resistin/IGF-1 ratio (but not IGF-1 alone) were inversely associated with quadriceps torque, while only adiponectin was inversely associated with handgrip strength independently from percentage of fat mass, height, age, gender and geographical origin. The ratio of leptin to adiponectin was directly associated with handgrip strength in both young and old subjects. These results suggest that in humans the age-associated loss of strength is associated with the levels of representative adipokines and IGF-1.

Abstracts of the 2023 Padua Days of Muscle and Mobility Medicine (2023Pdm3) to be held March 29 - April 1 at the Galileian Academy of Padua and at the Petrarca Hotel, Thermae of Euganean Hills, Padua, Italy.

At the end of the 2022 Padua Days of Muscle and Mobility Medicine (Pdm3) the next year's meeting was scheduled from 29 March to 1 April 2023. Despite the worsening evolution of the crisis in Eastern Europe, the program was confirmed in autumn 2022 with Scientific Sessions that will take place over three full days in the Aula Guariento of the Galileian Academy of Arts, Letters and Sciences of Padua (March 29, 2023) and then at the Conference Room of the Hotel Petrarca, Thermae of Euganean Hills (Padua), Italy. Collected during autumn and early winter, many titles and abstracts where submitted (about 100 Oral presentations are listed in the preliminary Program by January 31, 2023) confirming attractiveness of the 2023 Pdm3. The four days will include oral presentations of scientists and clinicians from Austria, Bulgaria, Canada, Denmark, France, Georgia, Germany, Iceland, Ireland, Italy, Mongolia, Norway, Russia, Slovakia, Slovenia, Spain, Switzerland, The Netherlands and USA. Together with the preliminary Program at January 31, 2023, the Collection of Abstracts is e-published in this Issue 33 (1) 2023 of the European Journal of Translational Myology (EJTM). You are invited to join, submitting your Last Minute Abstracts to ugo.carraro@unipd.it by March 15, 2023. Furthermore, with the more generous deadline of May 20, 2023, submit please "Communications" to the European Journal of Translational Myology (SCOPUS Cite Score Tracker 2023: 3.2 by January 5, 2023) and/or to the 2023 Special Issue: "Pdm3" of the Journal Diagnostics, MDPI, Basel (I.F. near to 4.0) with deadline September 30, 2023. Both journals will provide discounts to the first accepted typescripts. See you soon at the Hotel Petrarca of Montegrotto Terme, Padua, Italy. For a promo of the 2023 Pdm3 link to: https://www.youtube.com/watch?v=zC02D4uPWRg.

Loss of neuromuscular junction integrity and muscle atrophy in skeletal muscle disuse.

Physical inactivity (PI) is a major risk factor of chronic diseases. A major aspect of PI is loss of muscle mass and strength. The latter phenomenon significantly impacts daily life and represent a major issue for global health. Understandably, skeletal muscle itself has been the major focus of studies aimed at understanding the mechanisms underlying loss of mass and strength. Relatively lesser attention has been given to the contribution of alterations in somatomotor control, despite the fact that these changes can start very early and can occur at multiple levels, from the cortex down to the neuromuscular junction (NMJ). It is well known that exposure to chronic inactivity or immobilization causes a disproportionate loss of force compared to muscle mass, i.e. a loss of specific or intrinsic whole muscle force. The latter phenomenon may be partially explained by the loss of specific force of individual muscle fibres, but several other players are very likely to contribute to such detrimental phenomenon. Irrespective of the length of the disuse period, the loss of force is, in fact, more than two-fold greater than that of muscle size. It is very likely that somatomotor alterations may contribute to this loss in intrinsic muscle force. Here we review evidence that alterations of one component of somatomotor control, namely the neuromuscular junction, occur in disuse. We also discuss some of the novel players in NMJ stability (e.g., homer, bassoon, pannexin) and the importance of new established and emerging molecular markers of neurodegenerative processes in humans such as agrin, neural-cell adhesion molecule and light-chain neurofilaments.

Pathophysiological mechanisms of reduced physical activity: Insights from the human step reduction model and animal analogues.

Physical inactivity represents a heavy burden for modern societies and is spreading worldwide, it is a recognised pandemic and is the fourth cause of global mortality. Not surprisingly, there is an increasing interest in longitudinal studies on the impact of reduced physical activity on different physiological systems. This narrative review focuses on the pathophysiological mechanisms of step reduction (SR), an experimental paradigm that involves a sudden decrease in participants' habitual daily steps to a lower level, mimicking the effects of a sedentary lifestyle. Analogous animal models of reduced physical activity, namely, the "wheel-lock" and the "cage reduction" models, which can provide the foundation for human studies, are also discussed. The empirical evidence obtained thus far shows that even brief periods of reduced physical activity can lead to substantial alterations in skeletal muscle health and metabolic function. In particular, decrements in lean/muscle mass, muscle function, muscle protein synthesis, cardiorespiratory fitness, endothelial function and insulin sensitivity, together with an increased fat mass and inflammation, have been observed. Exercise interventions seem particularly effective for counteracting these pathophysiological alterations induced by periods of reduced physical activity. A direct comparison of SR with other human models of unloading, such as bed rest and lower limb suspension/immobilisation, is presented. In addition, we propose a conceptual framework aiming to unravel the mechanisms of muscle atrophy and insulin resistance in the specific context of reduced ambulatory activity. Finally, methodological considerations, knowledge gaps and future directions for both animal and human models are also discussed in the review.