Unilateral Lower Limb Suspension Research Articles

Resistance exercise following short term muscle disuse leads to enhanced mitochondrial activity and dynamics

Introduction: Short-term muscle unloading (~7-10 days) can be usually experienced following injury or hospitalisation, leading to muscle disuse atrophy, muscle weakness and changes in motor control. In such contexts, exercise countermeasures can be employed to overcome these neuromuscular impairments. Surprisingly, the effects of exercise countermeasures following the exposure to muscle unloading has been scarcely investigated. Hence, this study aimed to investigate the functional and molecular adaptations to a 21-day resistance training (RT) program following short term muscle disuse. We hypothesised that a period of resistance exercise following short-term unloading would speed up the recovery on muscle mass and function mostly by an effcient counteraction of the molecular regulators of muscle disuse atrophy consistent with the exercise modality employed. Methods: A 10-day unilateral lower limb suspension (ULLS) model was applied to eleven healthy young males (22.09 ± 2.91 y), followed by a 21-day RT program for the knee extensors performed at 70% of the 1RM (3 times/wk, 9 sessions in total). Data acquisition was performed before the ULLS period, after 10 days of unloading and after 21 days of resistance exercise. Quadriceps femoris isometric maximum voluntary contraction (MVC) was evaluated by dynamometry and quadriceps CSA was assessed by ultrasonography. Succinate dehydrogenase (SDH, a key component of the citric acid cycle and of the respiratory electron transfer chain) staining was performed on frozen cryosections obtained from VL biopsies, whereas western blot analyses were carried out for several mitochondrial-related proteins and transcriptomics data were obtained by RNA-seq. Results: Following ULLS, QF MVC and CSA decreased (-29.3%, p<0.001, and -4.5%, p<0.05, respectively). After the 21-d of RT, MVC was fully brought back (+42% compared to post-ULLS) whereas QF CSA showed a very marked hypertrophy (+18.6%, p<0.001, compared to post-ULLS). SDH immunoreactivity was not affected by unloading but revealed a significant increase after exercise compared to post ULLS values (~+20%, p<0.001). In addition, mitochondrial dynamics were highly influenced by resistance exercise, as mitochondrial pro-fusion (MFN1, MFN2, OPA1) and fission (DRP1, pDRP1616) proteins were markedly increased in response to RT compared to post-ULLS values (p<0.05). Further, PGC-1a levels showed a trend to decrease after ULLS but even a stronger trend to increase after resistance exercise compared to post-ULLS (p=0.056). Transcriptomics analyses showed that the most differentially expressed genes were identified in the pathways related to oxidative phosphorylation, in which the top regulated genes after resistance training were mitochondrial related genes (CKMT2, UQCRH, ACSS1, MDH1, COX7A2, CYCS, SLC16A1), presenting a marked reduction after ULLS and being significantly enhanced after RT. When compared to baseline, the genes which showed a positive “rebound effect” after resistance exercise were mostly related to mitochondrial function (MT-ATP6, MT-CO2, MT-ND3). Noteworthy, among these latter genes, the top upregulated one was APLNR, coding for apelin receptor protein, a positive regulator of inter-myofibrillar mitochondrial content in sarcopenia and ageing. Conclusions: Resistance exercise was effective in recovering muscle mass and function after 10 days of disuse, but it enhanced mitochondrial activity, biogenesis, and dynamics far beyond the expected effects for such exercise modality. In fact, mitochondrial-related genes expression was the most affected by exercise. This suggests that mitochondrial dynamics could play an essential role in the recovery of neuromuscular alterations induced by short term disuse. We put forward the hypothesis that the prior level of physical activity and a prior exposure to short-term muscle disuse could be driving the nature of adaptations to subsequent resistance training. Funding: The present work was funded by the Italian Space Agency (ASI), MARS-PRE, Project, n. DC-VUM-2017-006. This is the full abstract presented at the American Physiology Summit 2024 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.

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

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 PICs, contributing to the reduction of force generation capacity. Additionally, we tested whether physical exercise would restore the force generation capacity by re-establishing the initial level of neuromodulatory input. In 12 young adults, we assessed maximal voluntary contraction (MVC) pre- and post- 10 days of ULLS and following 21 days 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 (∆F) of paired motor units calculated during isometric ramped contractions. The values of ∆F were reduced after 10 days of ULLS (-33%, p < 0.001), but were fully re-established 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 MVC after ULLS (-29%, p = 0.002) and its recovery after the AR (+28.5%, p = 0.003). 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.

Neuromodulatory contribution to muscle force is reduced after 10 days of unilateral lower limb suspension

Neuromodulatory contribution to muscle force is reduced after 10 days of unilateral lower limb suspension

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

PurposeThis 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). MethodsTen 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. ResultsWe 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. ConclusionOur 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.

Upregulation of Sarcolemmal Hemichannels and Inflammatory Transcripts with Neuromuscular Junction Instability during Lower Limb Unloading in Humans.

Human skeletal muscle atrophy and a disproportionate force loss occur within a few days of unloading in space and on Earth, but the underlying mechanisms are not fully understood. Disruption of neuromuscular junction homeostasis has been proposed as one of the possible causes. Here, we investigated the potential mechanisms involved in this neuromuscular disruption induced by a 10-day unilateral lower limb suspension (ULLS) in humans. Specifically, we investigated hemichannels' upregulation, neuromuscular junction and axonal damage, neurotrophins' receptor downregulation and inflammatory transcriptional signatures. Biomarkers were evaluated at local and systemic levels. At the sarcolemmal level, changes were found to be associated with an increased expression of connexin 43 and pannexin-1. Upregulation of the inflammatory transcripts revealed by deep transcriptomics was found after 10 days of ULLS. The destabilisation of the neuromuscular junction was not accompanied by changes in the secretion of the brain-derived neurotrophic factor and neurotrophin-4, while their receptor, BDNF/NT growth factors receptor (TrkB), decreased. Furthermore, at 5 days of ULLS, there was already a significant upregulation of the serum neurofilament light chain concentration, an established clinical biomarker of axonal injury. At 10 days of ULLS, other biomarkers of early denervation processes appeared. Hence, short periods of muscle unloading induce sarcolemmal hemichannels upregulation, inflammatory transcripts upregulation, neuromuscular junction instability and axonal damage.

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 21days 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 21days 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.

Disuse Atrophy Elevates Skeletal Muscle CD63 Expression and Ex Vivo Release of Extracellular Vesicles in a Muscle‐Specific Manner

Extracellular vesicles (EVs) are recognized as mediators of intercellular communication during skeletal muscle adaptation to various stimuli. The role and behavior of skeletal-muscle derived EVs during disuse atrophy, however, has not been well characterized. The purpose of this study was to determine how disuse atrophy alters the release of muscle-derived EVs and well-known factors associated with EV production in rat and human muscle. Adult 10 month old male Fisher 344/BN rats underwent control, weight-bearing conditions (WB; n=7) or hindlimb suspension (HS) for either 4 hours (4h HS; n=4), 24 hours (24h HS; n=4), or 7 days (7d HS; n=5). Muscle-derived EVs were collected by excising the soleus muscle and incubating it in continuously gassed (95% O2/5% CO2) Kreb's Henseleit Buffer (118.5mM NaCl, 1.2mMMgSO4, 4.7mM KCl, 1.2mM KH2PO4, 25mM NaHCO3, 2.5mM CaCl2; pH 7.4) supplemented with 5mM glucose at 37OC for 1 hour. The soleus muscle was chosen for ex vivo experiments since it atrophies extensively and can be excised without disrupting the myofibers. EV abundance in the Kreb's buffer was measured using a Zetaview® Nanoparticle Tracking Analyzer. Skeletal muscle biopsies of the vastus lateralis (VL) were obtained from human subjects before and after 7 days of unilateral lower limb suspension (ULLS). RT-qPCR analysis was used to measure mRNA expression of factors in the soleus (rat), gastrocnemius (rat), and VL (human) muscles that are associated with EV production, including CD63, CD9, RAB27A, RAB27B, SMPD3, ARF6, CTTN, ANXA2, ISG15, and ARRDC1. We further examined CD63 protein expression via immunohistochemical (IHC) analysis. Muscle-derived EV release/mg muscle was 28% higher from 4h HS and 66% and 60% higher from 24h and 7d HS muscle, respectively, compared with WB (p<0.05 for both 4h HS and 24h HS). In addition, CD63, which is both a marker for and can mediate production of EVs, mRNA expression was positively correlated with ex-vivo skeletal muscle EV release (R2=0.22, P=0.03). Interestingly, CD63 mRNA expression, as well as expression of other EV factors, did not follow the same pattern of changes in the gastrocnemius suggesting muscle-specific changes in EV biology with disuse atrophy. Most EV factors had lower mRNA expression in the 4h HS and 24h HS groups with only RAB27A and RAB27B remaining lower expression in the 7dHS gastrocnemius muscle. However, CD63 expression was significantly increased in the VL from human subjects after disuse atrophy similar to the soleus from rats. IHC analysis of the soleus showed that the presence of CD63 protein was exclusive to a subset of non-myocyte cells within skeletal muscle. Altogether, our data demonstrate that disuse atrophy can increase CD63 expression and muscle EV release, although this may be in a muscle-specific or fiber type-specific manner. In addition, the majority of the change in EV production with disuse atrophy may be dictated by non-myocyte cells since there is no detection of CD63 in myocytes and CD63 protein is in very high abundance only in a subset of cells residing in the muscle interstitial space.

Serum extracellular vesicle miR-203a-3p content is associated with skeletal muscle mass and protein turnover during disuse atrophy and regrowth.

Small noncoding microRNAs (miRNAs) are important regulators of skeletal muscle size, and circulating miRNAs within extracellular vesicles (EVs) may contribute to atrophy and its associated systemic effects. The purpose of this study was to understand how muscle atrophy and regrowth alter in vivo serum EV miRNA content. We also associated changes in serum EV miRNA with protein synthesis, protein degradation, and miRNA within muscle, kidney, and liver. We subjected adult (10 mo) F344/BN rats to three conditions: weight bearing (WB), hindlimb suspension (HS) for 7 days to induce muscle atrophy, and HS for 7 days followed by 7 days of reloading (HSR). Microarray analysis of EV miRNA content showed that the overall changes in serum EV miRNA were predicted to target major anabolic, catabolic, and mechanosensitive pathways. MiR-203a-3p was the only miRNA demonstrating substantial differences in HS EVs compared with WB. There was a limited association of EV miRNA content to the corresponding miRNA content within the muscle, kidney, or liver. Stepwise linear regression demonstrated that EV miR-203a-3p was correlated with muscle mass and muscle protein synthesis and degradation across all conditions. Finally, EV miR-203a-3p expression was significantly decreased in human subjects who underwent unilateral lower limb suspension (ULLS) to induce muscle atrophy. Altogether, we show that serum EV miR-203a-3p expression is related to skeletal muscle protein turnover and atrophy. We suggest that serum EV miR-203a-3p content may be a useful biomarker and future work should investigate whether serum EV miR-203a-3p content is mechanistically linked to protein synthesis and degradation.

The Efficacy Of Protein Supplementation On Attenuating Muscle Atrophy Following Disuse

PURPOSE: The purpose of this study was to determine the effects of protein supplementation during muscular disuse in maintaining muscle cross-sectional area, lean mass, strength, and jump force in the collegiate population. METHODS: Two groups of healthy collegiate participants (n=14) underwent two weeks of unilateral lower limb suspension (ULLS), during which the control group consumed a normal diet (CON) and the treatment group (PRO) received an additional 75g/day of supplemental protein. Outcome measures included lean mass (LM) measured using DXA, muscle area (MA) of the lower leg using pQCT, isokinetic torque measurements using a Biodex ergometer, and force production during jumping using a portable force platform. Differences in the change of these outcome measurements from baseline to follow-up were compared between the treatment and control groups (group ∗ time interaction, controlling for baseline measurement). RESULTS: Daily protein intake for the treatment group (2.6±1.0 g/kg/d) was greater than the control group (1.5±1.1 g/kg/d). Daily caloric intake was not different between groups (3015±1098 kcal/day; Treatment 2772±432 kcal/day). MA decreased in both groups following ULLS with no differences between groups measured. However, there was a moderate difference in change between groups with the PRO maintaining their total LM (group∗time interaction, p=0.08) and leg LM (group∗time interaction, p=0.1) when compared to the CON. CONCLUSION: This study demonstrates a universal loss in lower leg muscle area muscle during a relatively short period of disuse. Additionally, there was evidence of a trend toward a positive effect of protein supplementation on lean mass maintenance during disuse. The results of this study serve as an important first step of identifying nutritional interventions to augment rehabilitation.

One-leg inactivity induces a reduction in mitochondrial oxidative capacity, intramyocellular lipid accumulation and reduced insulin signalling upon lipid infusion: a human study with unilateral limb suspension

Aims/hypothesisPhysical inactivity, low mitochondrial function, increased intramyocellular lipid (IMCL) deposition and reduced insulin sensitivity are common denominators of chronic metabolic disorders, like obesity and type 2 diabetes. Yet, whether low mitochondrial function predisposes to insulin resistance in humans is still unknown.MethodsHere we investigated, in an intervention study, whether muscle with low mitochondrial oxidative capacity, induced by one-legged physical inactivity, would feature stronger signs of lipid-induced insulin resistance. To this end, ten male participants (age 22.4 ± 4.2 years, BMI 21.3 ± 2.0 kg/m2) underwent a 12 day unilateral lower-limb suspension with the contralateral leg serving as an active internal control.ResultsIn vivo, mitochondrial oxidative capacity, assessed by phosphocreatine (PCr)-recovery half-time, was lower in the inactive vs active leg. Ex vivo, palmitate oxidation to 14CO2 was lower in the suspended leg vs the active leg; however, this did not result in significantly higher [14C]palmitate incorporation into triacylglycerol. The reduced mitochondrial function in the suspended leg was, however, paralleled by augmented IMCL content in both musculus tibialis anterior and musculus vastus lateralis, and by increased membrane bound protein kinase C (PKC) θ. Finally, upon lipid infusion, insulin signalling was lower in the suspended vs active leg.Conclusions/interpretationTogether, these results demonstrate, in a unique human in vivo model, that a low mitochondrial oxidative capacity due to physical inactivity directly impacts IMCL accumulation and PKCθ translocation, resulting in impaired insulin signalling upon lipid infusion. This demonstrates the importance of mitochondrial oxidative capacity and muscle fat accumulation in the development of insulin resistance in humans.Trial registrationClinicalTrial.gov NCT01576250.FundingPS was supported by a ‘VICI’ Research Grant for innovative research from the Netherlands Organization for Scientific Research (Grant 918.96.618).

Lower Loading Schemes Show Higher Power Generation and Activation of Select Muscles During Exercise on a Gravity‐Independent Resistance Device

Exposure to microgravity in space has resulted in reduced muscle size and strength. The YoYo™ inertial flywheel device presents a gravity‐independent method to counteract effects of deconditioning due to microgravity exposure. Previous studies utilizing flywheel devices have been shown to induce muscle size and strength during ambulation, unilateral lower limb suspension, and bed rest. Research examining optimal exercise prescription using flywheel devices is lacking. With the growing population of female astronauts, the purpose of this study was to explore the effects of squatting with various amounts of flywheel resistance on electromyography (EMG) of the leg musculature to more effectively guide the prescription of exercise regimens using flywheel devices. Eleven recreationally trained females who have exercised regularly for 6 months prior (20.2 ±1.6 yrs, 167 ±6.8 cm, 133.32 ±7.5 kg.) participated in the study. Participants conducted four randomized sets of seven repetitions of the squat exercise at different loading schemes. Loads were determined by interchanging and/or combining flywheel disks of two different moments of inertia: #1 disk (0.025 Kg*m2 moment of inertia) and #2 (0.050 Kg*m2) into four different loading schemes: #1 disk (0.025 Kg*m2), #2 disk (0.050 Kg*m2 m), #1, #2 disks (0.075 Kg*m2 m), and two #2 disks (0.100 Kg*m2 m). Average and peak power generation (W) and muscle electrical activity (uV) of the vastus medialis (VM), vastus lateralis (VL), rectus femoris (RF), biceps femoris (BF), semitendinosus (ST), and gluteus maximus (GM) were measured utilizing Bluebrain software and a Noraxon DTS Desktop EMG system. A linear mixed‐effects model was used to determine the effects of load and repetition on the dependent variables with significance set at p ≤ 0.05. Analysis revealed a significant main effect for load (p &lt; 0.001) and repetition (p &lt; 0.001) with the #1 disk showing the highest power output and a general increase in power output as exercise sets progressed (repetitions increased). Load showed a trend (p = 0.052) for peak EMG activity of the RF with Disk #2 having the highest peak EMG and increasing EMG activity as sets progressed (rep main effect, p = 0.002). Mean EMG activity was affected by load only for the BF (p = 0.014) with a trend for the GM (p = 0.058) both showing highest mean activity during the #1 disk set. In conclusion, the YoYo™ flywheel may be more effective in targeting specific muscles at lower loading schemes due to higher activations of muscle. Power output was consistently higher at lower loading schemes, suggesting more effective usage at lower loading schemes with the unique eccentric overload that occurs with this device. Results suggest possibilities of prescribing exercise on the flywheel device utilizing lower inertial loads but also highlight a need to explore additional parameters such as number of sets and repetitions needed to optimize chronic adaptations to exercise on a flywheel device.Support or Funding InformationThis research was supported by the National Institutes of Health under Award Numbers; 5UL1GM118979; 5TL4GM118980; 5RL5GM118978.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects Of Concurrent Exercise During Simulated Microgravity On Soleus Muscle Fiber Myonuclear Content

INTRODUCTION: Exercise countermeasures administered on the International Space Station are time-consuming and use large/expensive equipment. Aerobic exercise has shown to maintain oxidative capacity of muscle fibers, while resistance exercise preserves muscle mass in Astronauts. Recently, Cotter et al. (2015) determined that concurrent exercise (combining aerobic and resistance exercises; CE) was effective at mitigating de-conditioning during ULLS (unilateral lower limb suspension, simulated microgravity) but resulted in differences in fiber-type responses (i.e., fast-twitch vs slow-twitch). This current study provides additional analyses of muscle samples used in Cotter et al. (2015), investigating soleus muscle (mostly slow-twitch) myonuclei. Modulations in fiber size and myonuclear domain (MND; the area each myonuclei controls) may provide mechanisms for preventing unloading decrements. PURPOSE: Our aim was to determine 1) if simulated microgravity affects soleus muscle fiber size and MND size and 2) if CE training mitigates these changes. Sixteen subjects were separated into two groups, 10-day ULLS and 10-day ULLS + CE. METHODS: Muscle biopsies were taken pre- and post- intervention, which were isolated into individual fibers (muscle cells), stained for myonuclei, three-dimensionally imaged, and analyzed for fiber size and MND size. 2x2 ANOVAs determined potential differences in fiber size and MND size between groups, before and after ULLS. RESULTS: No significant differences in fiber size or MND size after 10 days of ULLS were observed. These findings suggest that, while 10 days of ULLS may cause a decline in muscle function (as seen in Cotter et al., 2015), it may not significantly affect soleus muscle fiber size or MND size. However, individual variability occurred, suggesting that some people may be responders (or non-responders) to ULLS and ULLS+CE. CONCLUSION: The results of this study suggest a need for additional analyses to help develop “personalized” exercise countermeasures for those undergoing significant periods of unloading (e.g., people in bed rest or Astronauts).

Disuse skeletal muscle atrophy in humans. Proteomic and molecular adaptations

Disuse skeletal muscle atrophy in humans. Proteomic and molecular adaptations

Relationship of changes in strain rate indices estimated from velocity-encoded MR imaging to loss of muscle force following disuse atrophy.

This study explores changes in strain rate (SR) (rate of regional deformation) parameters extracted from velocity-encoded MRI and their relationship to muscle force loss following 4-week unilateral lower limb suspension in healthy humans. Two-dimensional SR maps were derived from three directional velocity-encoded MR phase-contrast images of the medial gastrocnemius in seven subjects. Atrophy-related and regional differences in the SR eigenvalues, angle between the SR and muscle fiber (SR-fiber angle), and strain rates in the fiber basis were statistically analyzed using analysis of variance and linear regression. During isometric contraction, SR in the fiber cross section (SRin-plane ) was significantly lower, and the SR-fiber angle was significantly higher postsuspension (P < 0.05). On multiple variable regression analysis, the volume of medial gastrocnemius, SRin-plane , and SR-fiber angle were significantly associated with force and changes in the, and the SR eigenvalues and shear SR were significantly associated with change in force with disuse. Changes in SR-fiber angle, SRin-plane , and shear SR as well as their ability to predict force and force changes may reflect the role of remodeling of the extracellular matrix in disuse atrophy and its functional consequence in reducing lateral transmission of force. Magn Reson Med 79:912-922, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Dissociation between short-term unloading and resistance training effects on skeletal muscle Na+,K+-ATPase, muscle function, and fatigue in humans.

Physical training increases skeletal muscle Na+,K+-ATPase content (NKA) and improves exercise performance, but the effects of inactivity per se on NKA content and isoform abundance in human muscle are unknown. We investigated the effects of 23-day unilateral lower limb suspension (ULLS) and subsequent 4-wk resistance training (RT) on muscle function and NKA in 6 healthy adults, measuring quadriceps muscle peak torque; fatigue and venous [K+] during intense one-legged cycling exercise; and skeletal muscle NKA content ([3H]ouabain binding) and NKA isoform abundances (immunoblotting) in muscle homogenates (α1-3, β1-2) and in single fibers (α1-3, β1). In the unloaded leg after ULLS, quadriceps peak torque and cycling time to fatigue declined by 22 and 23%, respectively, which were restored with RT. Whole muscle NKA content and homogenate NKA α1-3 and β1-2 isoform abundances were unchanged with ULLS or RT. However, in single muscle fibers, NKA α3 in type I (-66%, P = 0.006) and β1 in type II fibers (-40%, P = 0.016) decreased after ULLS, with other NKA isoforms unchanged. After RT, NKA α1 (79%, P = 0.004) and β1 (35%, P = 0.01) increased in type II fibers, while α2 (76%, P = 0.028) and α3 (142%, P = 0.004) increased in type I fibers compared with post-ULLS. Despite considerably impaired muscle function and earlier fatigue onset, muscle NKA content and homogenate α1 and α2 abundances were unchanged, thus being resilient to inactivity induced by ULLS. Nonetheless, fiber type-specific downregulation with inactivity and upregulation with RT of several NKA isoforms indicate complex regulation of muscle NKA expression in humans.

Blood Flow Restricted Exercise Compared to High Load Resistance Exercise During Unloading.

Bed rest studies have shown that high load (HL) resistance training can mitigate the loss of muscle size and strength during musculoskeletal unloading; however, not all individuals are able to perform HL resistance exercise. Blood flow restricted (BFR) resistance exercise may be a novel way to prevent maladaptation to unloading without requiring HL exercise equipment. This study evaluated the muscular training adaptations to HL and BFR resistance training during unilateral lower limb suspension (ULLS), a human limb unloading model. ULLS allows for evaluation of exercise training in both weight-bearing and nonweight-bearing legs within the same individual. There were 13 participants who completed 25 d of ULLS and were counterbalanced to: 1) HL, N = 6; or 2) BFR, N = 7, training groups. During ULLS, HL and BFR performed unilateral leg press and heel raise exercise (3 d/wk). In weight-bearing legs, both HL and BFR increased knee extensor muscle cross-sectional area (CSA) and strength. In nonweight-bearing legs, knee extensor CSA and strength increased only in HL and decreased with BFR. HL and BFR resistance exercise were both effective exercise programs for the weight-bearing leg. However, BFR exercise was not as effective as HL resistance exercise in the nonweight-bearing leg. These data show that exercise that improved muscle CSA and strength in ambulatory weight-bearing conditions was not sufficient to maintain muscle function during unloading. For the preservation of muscle CSA and strength, BFR exercise should be considered an adjunct but not a primary exercise countermeasure for future space missions. Hackney KJ, Downs ME, Ploutz-Snyder L. Blood flow restricted exercise compared to high load resistance exercise during unloading. Aerosp Med Hum Perform. 2016; 87(8):688-696.

Effect of 23-day muscle disuse on sarcoplasmic reticulum Ca2+ properties and contractility in human type I and type II skeletal muscle fibers.

Inactivity negatively impacts on skeletal muscle function mainly through muscle atrophy. However, recent evidence suggests that the quality of individual muscle fibers is also altered. This study examined the effects of 23 days of unilateral lower limb suspension (ULLS) on specific force and sarcoplasmic reticulum (SR) Ca(2+) content in individual skinned muscle fibers. Muscle biopsies of the vastus lateralis were taken from six young healthy adults prior to and following ULLS. After disuse, the endogenous SR Ca(2+) content was ∼8% lower in type I fibers and maximal SR Ca(2+) capacity was lower in both type I and type II fibers (-11 and -5%, respectively). The specific force, measured in single skinned fibers from three subjects, decreased significantly after ULLS in type II fibers (-23%) but not in type I fibers (-9%). Western blot analyses showed no significant change in the amounts of myosin heavy chain (MHC) I and MHC IIa following the disuse, whereas the amounts of sarco(endo)plasmic reticulum Ca(2+)-ATPase 1 (SERCA1) and calsequestrin increased by ∼120 and ∼20%, respectively, and the amount of troponin I decreased by ∼21%. These findings suggest that the decline in force and power occurring with muscle disuse is likely to be exacerbated in part by reductions in maximum specific force in type II fibers, and in the amount of releasable SR Ca(2+) in both fiber types, the latter not being attributable to a reduced calsequestrin level. Furthermore, the ∼3-wk disuse in human elicits change in SR properties, in particular a more than twofold upregulation in SERCA1 density, before any fiber-type shift.

Unilateral lower limb suspension: From subject selection to "omic" responses.

The unilateral lower limb suspension (ULLS) method was developed, introduced, and validated in the quest for a simple, effective, and highly reliable human analog to study the consequences of spaceflight on muscle size and function. Because withdrawal of weight bearing for no more than 2-3 days is sufficient to inflict disturbances in protein metabolism of postural muscles, it is imperative ULLS serves as a very powerful method to manifest skeletal muscle adaptations similar to those experienced in 0 g. Thus the rate of global muscle loss appears rather constant over the first 2 mo, amounting to about 2-3% per week. At the microscopic level, these changes are accompanied by a corresponding decrease in individual muscle fiber size. ULLS alters metabolism favoring more carbohydrate over fat substrate utilization. Altogether, these changes result in impaired work and endurance capacity of muscles being subjected to ULLS. Maximal voluntary force decreases out of proportion to the muscle loss, suggesting motor control is modified. Past reviews offer near exhaustive information on ULLS-induced responses with regard to the above changes. Hence, the current brief review describes more broadly the evolution of the ULLS model, from issues of subject recruitment and compliance control, to recent advances unraveling molecular mechanisms facilitating unloading-induced muscle wasting. Such knowledge is critical in designing future studies aimed at exploring and developing exercise countermeasures or other means to combat the debilitating effects on muscle experienced by astronauts during long-haul missions in Orbit.

Human skeletal muscle fibre contractile properties and proteomic profile: adaptations to 3 weeks of unilateral lower limb suspension and active recovery.

It is generally assumed that muscle fibres go through atrophy following disuse with a loss of specific force and an increase in unloaded shortening velocity. However, the underlying mechanisms remain to be clarified. Most studies have focused on events taking place during the development of disuse, whereas the subsequent recovery phase, which is equally important, has received little attention. Our findings support the hypotheses that the specific force of muscle fibres decreased following unilateral lower limb suspension (ULLS) and returned to normal after 3weeks of active recovery as a result of a loss and recovery of myosin and actin content. Furthermore, muscle fibres went through extensive qualitative changes in muscle protein pattern following ULLS, and these were reversed by active recovery. Resistance training was very effective in restoring both muscle mass and qualitative muscle changes, indicating that long-term ULLS did not prevent the positive effect of exercise on human muscle. Following disuse, muscle fibre function goes through adaptations such as a loss of specific force (PO /CSA) and an increase in unloaded shortening velocity, which could be a result of both quantitative changes (i.e. atrophy) and qualitative changes in protein pattern. The underlying mechanisms remain to be clarified. In addition, little is known about the recovery of muscle mass and strength following disuse. In the present study, we report an extensive dataset describing, in detail,the functional and protein content adaptations of skeletal muscle in response to both disuse and re-training. Eight young healthy subjects were subjected to 3weeks of unilateral lower limb suspension (ULLS), a widely used human model of disuse skeletal muscle atrophy. Needle biopsies samples were taken from the vastus lateralis muscle Pre-ULLS, Post-ULLS and after 3weeks of recovery during which heavy resistance training was performed. After disuse, cross-sectional area (CSA), PO /CSA and myosin concentration (MC) decreased in both type 1 and 2A skinned muscle fibres. After recovery, CSA and MC returned to levels comparable to those observed before disuse, whereas Po/CSA and unloaded shortening velocity reached a higher level. Myosin heavy chain isoform composition of muscle samples did not differ among the experimental groups. To study the mechanisms underlying such adaptations, a two-dimensional proteomic analysis was performed. ULLS induced a reduction of myofibrillar, metabolic (glycolytic and oxidative) and anti-oxidant defence system protein content. Resistance training was very effective in counteracting ULLS-induced alterations, indicating that long-term ULLS did not prevent the positive effect of exercise on human muscle.

Concurrent exercise on a gravity-independent device during simulated microgravity.

The objective of this study is to examine the effect of a high-intensity concurrent training program using a single gravity-independent device on maintaining skeletal muscle function and aerobic capacity during short-term unilateral lower limb suspension (ULLS). Nineteen subjects (10 males and 9 females; 21.0 ± 2.5 yr, 65.4 ± 12.2 kg) were separated into two groups: 1) 10-d ULLS only (n = 9) and 2) 10-d ULLS plus aerobic and resistance training (ULLS + EX, n = 10). Exercise was performed on a single gravity-independent Multi-Mode Exercise Device (M-MED) with alternating days of high-intensity interval aerobic training and maximal exertion resistance training. Aerobic capacity increased by 7% in ULLS + EX (P < 0.05). Knee extensor and ankle plantar flexor three-repetition maximum increased in the ULLS + EX group (P < 0.05), but this change was only different from ULLS in the plantar flexors (P < 0.05). Peak torque levels decreased with ULLS but were increased for the knee extensors and attenuated for the ankle plantar flexors with ULLS + EX (P < 0.05). A shift toward type IIx myosin heavy-chain mRNA occurred with ULLS and was reversed with ULLS + EX in the vastus lateralis (P < 0.05) but not the soleus. Myostatin and atrogin increased with ULLS in both the vastus lateralis and soleus, but this change was mitigated with ULLS + EX only in the vastus lateralis (P = 0.0551 for myostatin, P < 0.05 for atrogin). Citrate synthase was decreased in the soleus during ULLS but was increased with ULLS + EX (P < 0.05). These results indicate that an M-MED class countermeasure device appears to be effective at mitigating the deconditioning effects of microgravity simulated during a modified ULLS protocol.