Muscle Disuse Research Articles

Exercise mimicry: Characterization of nutraceutical agents that may contribute to mitochondrial homeostasis in skeletal muscle

While it is well established that endurance exercise improves mitochondrial health in skeletal muscle, optimizing nutraceutical agents to mimic these metabolic achievements in the cell remains a challenge. Nonetheless, the application of exercise mimetics is a fruitful direction to pursue, as they may target and activate the same mechanisms that are upregulated with exercise administration alone. This is particularly useful under conditions where contractile activity is compromised due to muscle disuse, disease, or aging. The agents Sulforaphane (SFN) and Urolithin A (UroA) represent our preliminary candidates for antioxidation and mitophagy, respectively, for maintaining mitochondrial turnover and homeostasis. SFN is a powerful inducer of the Nrf‐2‐ARE pathway, a mechanism that upregulates cellular defences against oxidative stress. On the other hand, the metabolite, UroA, has developed a reputable role in regulating mitochondrial turnover via mitophagy. The purpose of this ongoing study is to characterize these nutraceutical agents both in their time‐ and dose‐dependent capacities to induce changes in protein content relative to the antioxidant and mitophagy pathways, in C2C12 myotubes. Differentiated muscle cells were treated with two concentrations of each nutraceutical for 4 h, 24 h, or 48 h. Immunoblot analysis was conducted to measure changes in protein content. SFN treatment after 4 h rendered a marked increase in the master regulator for antioxidation, and transcription factor, Nrf‐2, with no apparent changes in its negative regulator Keap‐1 at any given time point. Nuclear‐cytoplasmic fractions confirmed Nrf‐2 translocation to the nucleus following 4 h of treatment, likely representing the potent activation of antioxidant genes. This aligns with the upregulation of the downstream antioxidant markers HO‐1 and NQO1, showing 4‐5‐fold increases as early as 4 h and 24 h, respectively, and maintained after 48 h. Despite modest effects with Urolithin A, some significant changes took place under basal conditions. The upstream kinase phospho‐AMPK was activated by 2‐fold as early as 4 hr of treatment with UroA, which subsided by 24 and 48 h. Previous reports observed changes with the agent on autophagy markers, which includes the modest 1.3‐fold increases in p62 and LC3‐II after 48 h of treatment. However, no observable changes took place with respect to the mitophagy markers phospho‐ULK1, Parkin, or PINK1. Nonetheless, our preliminary results suggest that these agents may be suitable candidates as exercise mimetics. These data also set the stage for an examination of the synergistic effect of these nutraceuticals, in combination with contractile activity, on mitochondrial turnover and function.

Mechanotherapy does not Enhance Recovery Following Muscle Disuse Atrophy in Female Rats

BackgroundRecovery from skeletal muscle atrophy is important for regaining function. We previously showed that mechanotherapy enhances regrowth of muscle in adult male rats by elevating protein synthesis.ObjectiveThe purpose of this study was to determine if female rats also respond to mechanotherapy during regrowth after disuse‐induced atrophy.HypothesisMechanotherapy in female rats augments muscle regrowth after atrophy by enhancing protein synthesis.MethodsAdult (10 month) F344/BN female rats were randomly assigned to one of four groups: weight‐bearing (WB; N=6), hindlimb suspension (HS; N=9), reambulation after HS without mechanotherapy (RA; N=9), or RA with mechanotherapy (RAM; N=10). HS lasted for 14 and RA for 7 days. Starting the day of RA, mechanotherapy was applied to the right gastrocnemius muscle at a load of 4.5N for 30 minutes every other day (4 bouts total). Rats received deuterium oxide (D2O) for determination of myofibrillar protein and RNA synthesis (Ksyn) and degradation (Kdeg). We analyzed gastrocnemius muscles for myonuclear number (dystrophin‐DAPI), satellite cell (SC) number (Pax7), and fiber type and cross‐sectional area (CSA; laminin‐myosin heavy chain). We used a one‐way ANOVA to determine statistical significance at p<0.05. Values are mean ± SD.ResultsMuscle wet weight (g) was lower with HS (933 ± 56) compared to WB (1140 ± 86) and greater in RA (1012 ± 64) and RAM (1030 ± 41) compared to HS. However, no difference was observed between RA and RAM. Mean fiber CSA (µm2) was significantly lower in HS (1877 ± 244) compared to WB (2395 ± 248), while RA (2124 ± 287) was not different from WB or HS. RAM CSA (2013 ± 182) was lower than WB and not different from HS or RA. Myofibrillar Ksyn (mg/day) was lower in HS (13 ± 2.1) compared to WB (22 ± 2.2). RA (32 ± 3.2) and RAM (30 ± 4.3) were higher than HS and WB, but no difference was observed between RA and RAM. Myofibrillar Kdeg (1/t) was greater in HS (0.03 ± 0.004) compared to WB (0.02 ± 0.005), and RA (0.04 ± 0.007) and RAM (0.04 ± 0.005) were higher than HS and WB, but not different from each other. RNA Ksyn (%/day) was not different between WB (2 ± 0.2) and HS (2 ± 0.3), whereas RE (3 ± 0.4) and REM (4 ± 0.6) were both higher that WB and HS, but not different from each other. There were no differences in fiber type distribution between the groups. CSA of all fiber types was lower with HS, whereas no differences were observed between RA, RAM and HS. We did not find a significant difference in myonuclear number or centrally located nuclei between the groups. The number of SCs (per/fiber) was lower in HS (0.03 ± 0.01) and RA (0.04 ± 0.01) compared to WB (0.05 ± 0.008) and was higher in RAM (0.05 ± 0.02) compared to HS.ConclusionMuscle recovery from atrophy in female rats occurs with reambulation and elevated protein turnover is the underlying anabolic mechanism. We therefore conclude that the additional load using mechanotherapy does not enhance muscle recovery in females, which is different than the response previously published for males.

Low-frequency electrical stimulation alleviates immobilization-evoked disuse muscle atrophy by repressing autophagy in skeletal muscle of rabbits

BackgroundThe study aimed to investigate the effect of low-frequency electrical stimulation (LFES) on disuse muscle atrophy and its mechanism in a rabbit model of knee extension contracture.MethodsThis study involved two experiments. In the time-point experiment, 24 rabbits were randomly divided into 4 groups: Control 1 (Ctrl1 group), immobilization for 2 weeks (I-2 group), immobilization for 4 weeks (I-4 group), and immobilization for 6 weeks (I-6 group). In the intervention experiment, 24 rabbits were randomly divided into 4 groups: Control 2 (Ctrl2 group), electrical stimulation (ESG group), natural recovery (NRG group), and electrical stimulation treatment (ESTG group). All intervention effects were assessed by evaluating the knee joint range of motion (ROM), cross-sectional area (CSA) of the rectus femoris muscle, and expression of autophagy-related proteins.ResultsThe time-point experiment showed that immobilization reduced the knee ROM, reduced the rectus femoris muscle CSA, and activated autophagy in skeletal muscle. The levels of five autophagy-related proteins [mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), autophagy-related protein 7 (Atg7), p62, and microtubule-associated protein light chain 3B-II (LC3B-II)] were significantly elevated in the skeletal muscle of the I-4 group. The intervention experiment further showed that LFES significantly improved the immobilization-induced reductions in ROM and CSA. Additionally, LFES resulted in a significant decrease in the protein expression of mTOR, p-mTOR, Atg7, p62, and LC3B-II in the rectus femoris muscle.ConclusionsLFES alleviates immobilization-evoked disuse muscle atrophy possibly by inhibiting autophagy in the skeletal muscle of rabbits.

Disuse-induced skeletal muscle atrophy in disease and nondisease states in humans: mechanisms, prevention, and recovery strategies.

Decreased skeletal muscle contractile activity (disuse) or unloading leads to muscle mass loss, also known as muscle atrophy. The balance between muscle protein synthesis (MPS) and muscle protein breakdown (MPB) is the primary determinant of skeletal muscle mass. A reduced mechanical load on skeletal muscle is one of the main external factors leading to muscle atrophy. However, endocrine and inflammatory factors can act synergistically in catabolic states, amplifying the atrophy process and accelerating its progression. In addition, older individuals display aging-induced anabolic resistance, which can predispose this population to more pronounced effects when exposed to periods of reduced physical activity or mechanical unloading. Different cellular mechanisms contribute to the regulation of muscle protein balance during skeletal muscle atrophy. This review summarizes the effects of muscle disuse on muscle protein balance and the molecular mechanisms involved in muscle atrophy in the absence or presence of disease. Finally, a discussion of the current literature describing efficient strategies to prevent or improve the recovery from muscle atrophy is also presented.

An Evidence-Based Narrative Review of Mechanisms of Resistance Exercise-Induced Human Skeletal Muscle Hypertrophy.

ABSTRACTSkeletal muscle plays a critical role in physical function and metabolic health. Muscle is a highly adaptable tissue that responds to resistance exercise (RE; loading) by hypertrophying, or during muscle disuse, RE mitigates muscle loss. Resistance exercise training (RET)–induced skeletal muscle hypertrophy is a product of external (e.g., RE programming, diet, some supplements) and internal variables (e.g., mechanotransduction, ribosomes, gene expression, satellite cells activity). RE is undeniably the most potent nonpharmacological external variable to stimulate the activation/suppression of internal variables linked to muscular hypertrophy or countering disuse-induced muscle loss. Here, we posit that despite considerable research on the impact of external variables on RET and hypertrophy, internal variables (i.e., inherent skeletal muscle biology) are dominant in regulating the extent of hypertrophy in response to external stimuli. Thus, identifying the key internal skeletal muscle–derived variables that mediate the translation of external RE variables will be pivotal to determining the most effective strategies for skeletal muscle hypertrophy in healthy persons. Such work will aid in enhancing function in clinical populations, slowing functional decline, and promoting physical mobility. We provide up-to-date, evidence-based perspectives of the mechanisms regulating RET-induced skeletal muscle hypertrophy.

A physical restraint device to facilitate rehabilitation in critically ill patients

Physical restraints are widely used in hospitalized and critically ill patients, especially in intensive care unit (ICU), to prevent adverse events such as the accidental removal of various monitoring leads, therapeutic tubes, and self-injury or injury to others due to delirium and irritation. The existing restraint measures directly bind the upper limbs of the patients to the hospital bed, which often brings psychological harm to the patients and leads to disuse muscular atrophy. Early rehabilitation therapy can help improve the prognosis of patients,but it is difficult to be widely used in ICU due to being heavily dependent on nursing and rehabilitation physicians. A novel restraint device to facilitate rehabilitation training for critically ill patients was designed by the medical staff from the department of critical care medicine of Beijing Tiantan Hospital, Capital Medical University and obtained the National Utility Model Patent of China (ZL 2020 2 2492749.6). The device is mainly composed of a cross beam and a locking device whose two ends are connected by a rocker arm, an upper limb stopper and an upper body stopper. The upper limb and body restraint provide restrictions on the movement of the head, the upper limb, and the upper body. The angle limiter prevents the patient from pulling out the treatment tube by himself, and at the same time retains the ability to grasp the crossbar and rotate it, and the sliding block further increases the activity space, to meet the exercise of the patient's upper limb muscle strength. Carrying out physical rehabilitation training as early as possible during ICU treatment can relieve the patient's resistance to passive restraint, reduce the incidence of disuse muscle atrophy, eliminate the potential hidden dangers of medical disputes, and ultimately improve the prognosis of patients.

Implication of Exercise for Healthy Longevity in Older People

As the aging population is increasing substantially worldwide, extending the healthy life expectancy of older people has become a challenging issue. The structure and function of skeletal muscle decline with age, and this change has been shown to be involved in the development of various adverse health outcomes. In recent years, sarcopenia, a disease that is associated with age-related skeletal muscle mass loss and muscle weakness, has been attracting attention, and disuse muscle atrophy is also an important condition. For these diseases, it is recommended to increase exercise and physical activity, especially resistance exercise. In addition to conducting further research, it is necessary to widely disseminate information regarding the significance of exercise and physical activity as cost-effective interventions to the general public as well as health care professionals.

Regulatory networks coordinating mitochondrial quality control in skeletal muscle

The adaptive plasticity of mitochondria within a skeletal muscle is regulated by signals converging on a myriad of regulatory networks that operate during conditions of increased (i.e., exercise) and decreased (inactivity, disuse) energy requirements. Notably, some of the initial signals that induce adaptive responses are common to both conditions, differing in their magnitude and temporal pattern, to produce vastly opposing mitochondrial phenotypes. In response to exercise, signaling to peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α (PGC-1α) and other regulators ultimately produces an abundance of high-quality mitochondria, leading to reduced mitophagy and a higher mitochondrial content. This is accompanied by the presence of an enhanced protein quality control system that consists of the protein import machinery as well chaperones and proteases termed the mitochondrial unfolded protein response (UPRmt). The UPRmt monitors intraorganelle proteostasis, and strives to maintain a mito-nuclear balance between nuclear- and mtDNA-derived gene products via retrograde signaling from the organelle to the nucleus. In addition, antioxidant capacity is improved, affording greater protection against oxidative stress. In contrast, chronic disuse conditions produce similar signaling but result in decrements in mitochondrial quality and content. Thus, the interactive cross talk of the regulatory networks that control organelle turnover during wide variations in muscle use and disuse remain incompletely understood, despite our improving knowledge of the traditional regulators of organelle content and function. This brief review acknowledges existing regulatory networks and summarizes recent discoveries of novel biological pathways involved in determining organelle biogenesis, dynamics, mitophagy, protein quality control, and antioxidant capacity, identifying ample protein targets for therapeutic intervention that determine muscle and mitochondrial health.

Mechanical unloading of 3D-engineered muscle leads to muscle atrophy by suppressing protein synthesis

Three-dimensional (3D)-engineered muscle is an useful approach to a more comprehensive understanding of molecular mechanisms underlying unloading-induced muscle atrophy. We investigated the effects of mechanical unloading on molecular muscle protein synthesis (MPS)- and muscle protein breakdown (MPB)-related signaling pathways involved in muscle atrophy in 3D-engineered muscle, and to better understand in vitro model of muscle disuse. The 3D-engineered muscle consisting of C2C12 myoblasts and type-1 collagen gel was allowed to differentiate for 2 wk and divided into three groups: 0 days of stretched-on control (CON), 2 and/or 7 days of stretched-on (ON), in which both ends of the muscle were fixed with artificial tendons, and the stretched-off group (OFF), in which one side of the artificial tendon was detached. Muscle weight (-38.1% to -48.4%), length (-67.0% to -73.5%), twitch contractile force (-70.5% to -75.0%), and myosin heavy chain expression (-32.5% to -50.5%) in the OFF group were significantly decreased on days 2 and 7 compared with the ON group (P < 0.05, respectively), despite that ON group was stable over time. Although determinative molecular signaling could not be identified, the MPS rate reflected by puromysin-labeled protein was significantly decreased following mechanical unloading (P < 0.05, -38.5% to -51.1%). Meanwhile, MPB, particularly the ubiquitin-proteasome pathway, was not impacted. Hence, mechanical unloading of 3D-engineered muscle in vitro leads to muscle atrophy by suppressing MPS, cell differentiation, and cell growth rather than the promotion of MPB.NEW & NOTEWORTHY Three-dimensional (3D)-engineered muscles have recently been shown to closely replicate the in vivo architecture. We found that mechanical unloading of 3D-engineered muscle led to muscle disuse atrophy accompanied by reduced functional properties and contractile protein expression via suppression of muscle protein synthesis. This novel model may improve the in vitro testing of modalities with the potential to reduce mechanical unloading-induced atrophy.

Disuse-associated loss of the protease LONP1 in muscle impairs mitochondrial function and causes reduced skeletal muscle mass and strength

Mitochondrial proteolysis is an evolutionarily conserved quality-control mechanism to maintain proper mitochondrial integrity and function. However, the physiological relevance of stress-induced impaired mitochondrial protein quality remains unclear. Here, we demonstrate that LONP1, a major mitochondrial protease resides in the matrix, plays a role in controlling mitochondrial function as well as skeletal muscle mass and strength in response to muscle disuse. In humans and mice, disuse-related muscle loss is associated with decreased mitochondrial LONP1 protein. Skeletal muscle-specific ablation of LONP1 in mice resulted in impaired mitochondrial protein turnover, leading to mitochondrial dysfunction. This caused reduced muscle fiber size and strength. Mechanistically, aberrant accumulation of mitochondrial-retained protein in muscle upon loss of LONP1 induces the activation of autophagy-lysosome degradation program of muscle loss. Overexpressing a mitochondrial-retained mutant ornithine transcarbamylase (ΔOTC), a known protein degraded by LONP1, in skeletal muscle induces mitochondrial dysfunction, autophagy activation, and cause muscle loss and weakness. Thus, these findings reveal a role of LONP1-dependent mitochondrial protein quality-control in safeguarding mitochondrial function and preserving skeletal muscle mass and strength, and unravel a link between mitochondrial protein quality and muscle mass maintenance during muscle disuse.

Factors mediating spaceflight-induced skeletal muscle atrophy.

Skeletal muscle atrophy is a well-known consequence of spaceflight. Because of the potential significant impact of muscle atrophy and muscle dysfunction on astronauts and their mission, a thorough understanding of the mechanisms of this atrophy and the development of effective countermeasures is critical. Spaceflight-induced muscle atrophy is similar to atrophy seen in many terrestrial conditions, and therefore our understanding of this form of atrophy may also contribute to the treatment of atrophy in humans on Earth. The unique environmental features humans encounter in space include the weightlessness of microgravity, space radiation, and the distinctive aspects of living in a spacecraft. The disuse and unloading of muscles in microgravity are likely the most significant factors that mediate spaceflight-induced muscle atrophy and have been extensively studied and reviewed. However, there are numerous other direct and indirect effects on skeletal muscle that may be contributing factors to the muscle atrophy and dysfunction seen as a result of spaceflight. This review offers a novel perspective on the issue of muscle atrophy in space by providing a comprehensive overview of the unique aspects of the spaceflight environment and the various ways in which they can lead to muscle atrophy. We systematically review the potential contributions of these different mechanisms of spaceflight-induced atrophy and include findings from both actual spaceflight and ground-based models of spaceflight in humans, animals, and in vitro studies.

Plantar stimulation prevents the decrease in fatigue resistance in rat soleus muscle under one week of hindlimb suspension

Support afferentation in recent years was shown to be a key physiological stimulus controlling postural muscle function, structure and phenotype. Lack of support afferentation under various types of muscle disuse leads to a decline of size and percentage of slow-type fatigue-resistant muscle fibers, which can negatively affect muscle performance and life quality. In this study we simulated support afferentation during rat hindlimb unloading and investigated its effect on postural soleus muscle functional properties and signaling. Plantar mechanical stimulation prevented the unloading-induced muscle fatigue increase, maintained the level of mitochondrial DNA copy number and the percent of slow-type muscle fibers and partially prevented the increase of CpG methylation in pgc 1α promoter region and decline in myonuclear content of several transcriptional activators of slow myosin and PGC1 α expression. So, support afferentation under hindlimb suspension leads to maintaining of a slow-twitch oxidative and fatigue-resistant soleus muscle fibers phenotype.

Time-dependent changes in autophagy, mitophagy and lysosomes in skeletal muscle during denervation-induced disuse.

Deficits in skeletal muscle mitochondrial content and quality are observed following denervation-atrophy. This is due to alterations in the biogenesis of new mitochondria as well as their degradation via mitophagy. The regulation of autophagy and mitophagy over the course of denervation (Den) remains unknown. Further, the time-dependent changes in lysosome content, the end-stage organelle for mitophagy, remain unexplored. Here, we studied autophagic as well as mitophagic pre-lysosomal flux in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria from rat muscle subjected to Den for 1, 3 or 7days. We also assessed flux at 1day post-denervation in transgenic mt-keima mice. Markers of mitochondrial content were reduced at 7days following Den, and Den further resulted in rapid decrements in mitochondrial respiration, along with increased ROS emission. Pre-lysosomal autophagy flux was upregulated at 1 and 3days post-Den but was reduced compared to time-matched sham-operated controls at 7days post-Den. Similarly, pre-lysosomal mitophagy flux was enhanced in SS mitochondria as early as 1 and 3days of Den but decreased in both SS and IMF subfractions following 7days of Den. Lysosome protein content and transcriptional regulators TFEB and TFE3 were progressively enhanced with Den, an adaptation designed to enhance autophagic capacity. However, evidence for lysosome dysfunction was apparent by 7days, which may limit degradation capacity. This may contribute to an inability to clear dysfunctional mitochondria and increased ROS signalling, thereby accelerating muscle atrophy. Thus, therapeutic targeting of lysosome function may help to maintain autophagy and muscle health during conditions of muscle disuse or denervation. KEY POINTS: Denervation is an experimental model of peripheral neuropathies as well as muscle disuse, and it helps us understand some aspects of the sarcopenia of ageing. Muscle disuse is associated with reduced mitochondrial content and function, leading to metabolic impairments within the tissue. Although the processes that regulate mitochondrial biogenesis are understood, those that govern mitochondrial breakdown (i.e. mitophagy) are not well characterized in this context. Autophagy and mitophagy flux, measured up to the point of the lysosome (pre-lysosomal flux rates), were increased in the early stages of denervation, along with mitochondrial dysfunction, but were reduced at later time points when the degree of muscle atrophy was highest. Denervation led to progressive increases in lysosomal proteins to accommodate mitophagy flux, yet evidence for lysosomal impairment at later stages may limit the removal of dysfunctional mitochondria, stimulate reactive oxygen species signalling, and reduce muscle health as denervation time progresses.

Intensive care unit-acquired weakness and the COVID-19 pandemic: A clinical review.

Patients with severe cases of coronavirus disease 2019 (COVID-19) often become critically ill requiring intensive care unit (ICU) management. These individuals are at risk for developing ICU-acquired weakness (ICUAW), a multifactorial condition in which polyneuropathy, myopathy, and/or disuse muscle atrophy result in motor weakness. This weakness is thought to contribute to the long-term functional disability frequently observed in survivors of critical illness. This review discusses the current evidence regarding the epidemiology, pathophysiology, evaluation, risk factors, and rehabilitation-specific management of ICUAW in patients with COVID-19. Because of the novelty of COVID-19, the exact prevalence of ICUAW is not well delineated among COVID-19 patients. However, ICUAW has been reported in this population with retrospective studies showing weakness occurring in up to 45.5% of patients with severe COVID-19. There are multiple risk factors for developing ICUAW among COVID-19 patients, including premorbid health status, sepsis, multiple organ failure, mechanical ventilation, immobilization, neuromuscular blockade, corticosteroid use, and glycemic control. ICUAW is more likely to occur after prolonged mechanical ventilation and long hospital stays and can be diagnosed with manual muscle and electrodiagnostic testing. Although the long-term sequela of COVID-19 after ICU stays is not fully studied, increasing evidence indicates significant risk for this population developing long-term functional impairments. Establishing postacute rehabilitation programs for COVID-19 survivors will be important for recovery of endurance, mobility, and function.

Effect of High-Protein Diets on Integrated Myofibrillar Protein Synthesis before Anterior Cruciate Ligament Reconstruction: A Randomized Controlled Pilot Study.

Increasing dietary protein intake during periods of muscle disuse may mitigate the resulting decline in muscle protein synthesis (MPS). The purpose of this randomized pilot study was to determine the effect of increased protein intake during periods of disuse before anterior cruciate ligament (ACL) reconstruction on myofibrillar protein synthesis (MyoPS), and proteolytic and myogenic gene expression. Six healthy, young males (30 ± 9 y) were randomized to consume a high-quality, optimal protein diet (OP; 1.9 g·kg−1·d−1) or adequate protein diet (AP; 1.2 g·kg−1·d−1) for two weeks before ACL reconstruction. Muscle biopsies collected during surgery were used to measure integrated MyoPS during the intervention (via daily deuterium oxide ingestion) and gene expression at the time of surgery. MyoPS tended to be higher, with a large effect size in OP compared to AP (0.71 ± 0.1 and 0.54 ± 0.1%·d−1; p = 0.076; g = 1.56). Markers of proteolysis and myogenesis were not different between groups (p > 0.05); however, participants with greater MyoPS exhibited lower levels of MuRF1 gene expression compared to those with lower MyoPS (r = −0.82, p = 0.047). The data from this pilot study reveal a potential stimulatory effect of increased daily protein intake on MyoPS during injury-mediated disuse conditions that warrants further investigation.

Automated cross-sectional analysis of trained, severely atrophied, and recovering rat skeletal muscles using MyoVision 2.0.

The number of myonuclei within a muscle fiber is an important factor in muscle growth, but its regulation during muscle adaptation is not well understood. We aimed to elucidate the time course of myonuclear dynamics during endurance training, loaded and concentric resistance training, and nerve silencing-induced disuse atrophy with subsequent recovery. We modified tibialis anterior muscle activity in free-living rats with electrical stimulation from implantable pulse generators, or with implantable osmotic pumps delivering tetrodotoxin (TTX) to silence the motor nerve without transection. We used the updated, automated software MyoVision to measure fiber-type-specific responses in whole tibialis anterior cross sections (∼8,000 fibers each). Seven days of continuous low-frequency stimulation (CLFS) reduced muscle mass (-12%), increased slower myosin isoforms and reduced IIX/IIB fibers (-32%), and substantially increased myonuclei especially in IIX/IIB fibers (55.5%). High-load resistance training (spillover) produced greater hypertrophy (∼16%) in muscle mass and fiber cross-sectional area (CSA) than low-load resistance training (concentric, ∼6%) and was associated with myonuclear addition in all fiber types (35%-46%). TTX-induced nerve silencing resulted in progressive loss in muscle mass, fiber CSA, and myonuclei per fiber cross section (-50.7%, -53.7%, and -40.7%, respectively, at 14 days). Myonuclear loss occurred in a fiber-type-independent manner, but subsequent recovery during voluntary habitual activity suggested that type IIX/IIB fibers contained more new myonuclei during recovery from severe atrophy. This study demonstrates the power and accuracy provided by the updated MyoVision software and introduces new models for studying myonuclear dynamics in training, detraining, retraining, repeated disuse, and recovery.NEW & NOTEWORTHY We introduce new models for studying fiber-type-specific myonuclear dynamics in muscle training, detraining, retraining, disuse, and recovery. We show that the various fiber types do not respond identically and that myonuclear number changes during adaptation. We also critically assess an updated version of MyoVision automated image analysis software to quantify whole muscle immunofluorescent microscopical images in a faster and less computer intensive manner. MyoVision remains open source and freely available with more user-controlled features.

Costly immunometabolic remodelling in disused muscle buildup through physical exercise.

The mechanisms underlying the immunometabolic disturbances during skeletal muscle atrophy caused by a plethora of circumstances ranging from hospitalization to spaceflight missions remain unknown. Here, we outline the possible pathways that might be dysregulated in such conditions and assess the potential of physical exercise to mitigate and promote the recovery of muscle morphology, metabolism and function after intervals of disuse. Studies applying exercise to attenuate disuse-induced muscle atrophy have shown a pivotal role of circulating myokines in the activation of anabolic signalling pathways. These muscle-derived factors induce accretion of contractile proteins in the myofibers, and at the same time decrease protein breakdown and loss. Regular exercise plays a crucial role in re-establishing adequate immunometabolism and increasing the migration and presence in the muscle of macrophages with an anti-inflammatory phenotype (M2) and T regulatory cells (Tregs) after disease-induced muscle loss. Additionally, the switch in metabolic pathways (glycolysis to oxidative phosphorylation [OXPHOS]) is important for achieving rapid metabolic homeostasis during muscle regeneration. In this review, we discuss the molecular aspects of the immunometabolic response elicited by exercise during skeletal muscle regeneration. There is not, nevertheless, consensus on a single optimal intensity of exercise required to improve muscle strength, mass and functional capacity owing to the wide range of exercise protocols studied so far. Despite the absence of agreement on the specific strategy, physical exercise appears as a powerful complementary strategy to attenuate the harmful effects of muscle disuse in different scenarios.

The impact of skeletal muscle disuse on distinct echo intensity bands: A retrospective analysis.

Echo intensity (EI) is a novel tool for assessing muscle quality. EI has traditionally been reported as the mean of the pixel histogram, with 0 and 255 arbitrary units (A.U.) representing excellent and poor muscle quality, respectively. Recent work conducted in youth and younger and older adults suggested that analyzing specific EI bands, rather than the mean, may provide unique insights into the effectiveness of exercise and rehabilitation interventions. As our previous work showed deterioration of muscle quality after knee joint immobilization, we sought to investigate whether the increase in EI following disuse was limited to specific EI bands. Thirteen females (age = 21 yrs) underwent two weeks of left knee immobilization and ambulated via crutches. B-mode ultrasonography was utilized to obtain images of the immobilized vastus lateralis. The percentage of the total number of pixels within bands of 0-50, 51-100, 101-150, 151-200, and 201-255 A.U. was examined before and after immobilization. We also sought to determine if further subdividing the histogram into 25 A.U. bands (i.e., 0-25, 26-50, etc.) would be a more sensitive methodological approach. Immobilization resulted in a decrease in the percentage of pixels within the 0-50 A.U. band (-3.11 ± 3.98%), but an increase in the 101-150 A.U. (2.94 ± 2.64%) and 151-200 A.U. (0.93 ± 1.42%) bands. Analyses of variance on the change scores indicated that these differences were large and significant (%EI0-50 vs. %EI101-150: p < .001, d = 1.243); %EI0-50 vs. %EI151-200: p = .043, d = 0.831). The effect size for the %EI51-100 versus %EI101-150 comparison was medium/large (d = 0.762), but not statistically significant (p = .085). Further analysis of the 25 A.U. bands indicated that the percentage of pixels within the 25-50 A.U. band decreased (-2.97 ± 3.64%), whereas the 101-125 (1.62 ± 1.47%) and 126-150 A.U. (1.18 ± 1.07%) bands increased. Comparison of the 50 A.U. and 25 A.U. band methods found that 25 A.U. bands offer little additional insight. Though studies are needed to ascertain the factors that may influence specific bands, changes in EI during muscle disuse are not homogeneous across the pixel histogram. We encourage investigators to think critically about the robustness of data obtained from EI histograms, rather than simply reporting the EImean value, in muscle quality research.

Time course of capillary regression and an expression balance between vascular endothelial growth factor-A and thrombospondin-1 in the soleus muscle of hindlimb unloaded rats.

Skeletal muscle capillaries regress with disuse; however, information on time-dependent changes in the expression of pro- and anti-angiogenic factors in disused muscle is limited. This study aimed to clarify time-dependent changes in skeletal muscle capillarization, pro-angiogenic vascular endothelial growth factor-A (VEGF-A), and anti-angiogenic thrombospondin-1 (TSP-1) in the soleus muscle of hindlimb unloaded rat. Eight-week-old male Sprague Dawley rats were randomly divided into four groups corresponding to different hindlimb unloading (HU) duration at 0, 1, 2, and 3wk. Muscle atrophy and capillary regression worsened in the soleus muscle with longer periods of HU. The VEGF-A protein expression level was lower at week 1 than at week 0. In addition, the value at week 3 was also lower than those at weeks 0, 1, and 2. The TSP-1 protein expression level was higher at week 1 than that at week 0 but was similar at weeks 2 and 3. Moreover, reactive oxygen species, assessed by dihydroethidium fluorescence intensity on cryosection, were higher at weeks 2 and 3 than that at week 0. Depending on the HU period, VEGF-A and TSP-1 showed different expression patterns. In the early HU phase, TSP-1 may play an important role in capillary regression. However, when HU extends for a longer period, decreased VEGF-A, and/or increased oxidative stress may be more involved in capillary regression.

Measurement of Protein Import Capacity of Skeletal Muscle Mitochondria.

Mitochondria are key metabolic and regulatory organelles that determine the energy supply as well as the overall health of the cell. In skeletal muscle, mitochondria exist in a series of complex morphologies, ranging from small oval organelles to a broad, reticulum-like network. Understanding how the mitochondrial reticulum expands and develops in response to diverse stimuli such as alterations in energy demand has long been a topic of research. A key aspect of this growth, or biogenesis, is the import of precursor proteins, originally encoded by the nuclear genome, synthesized in the cytosol, and translocated into various mitochondrial sub-compartments. Mitochondria have developed a sophisticated mechanism for this import process, involving many selective inner and outer membrane channels, known as the protein import machinery (PIM). Import into the mitochondrion is dependent on viable membrane potential and the availability of organelle-derived ATP through oxidative phosphorylation. Therefore its measurement can serve as a measure of organelle health. The PIM also exhibits a high level of adaptive plasticity in skeletal muscle that is tightly coupled to the energy status of the cell. For example, exercise training has been shown to increase import capacity, while muscle disuse reduces it, coincident with changes in markers of mitochondrial content. Although protein import is a critical step in the biogenesis and expansion of mitochondria, the process is not widely studied in skeletal muscle. Thus, this paper outlines how to use isolated and fully functional mitochondria from skeletal muscle to measure protein import capacity in order to promote a greater understanding of the methods involved and an appreciation of the importance of the pathway for organelle turnover in exercise, health, and disease.