Identifying the potential therapeutic effects of miR­6516 on muscle disuse atrophy.

Muscle atrophy is a debilitating condition with various causes; while aging is one of these causes, reduced engagement in routine muscle­strengthening activities also markedly contributes to muscle loss. Although extensive research has been conducted on microRNAs (miRNAs/miRs) and their associations with muscle atrophy, the roles played by miRNA precursors remain underexplored. The present study detected the upregulation of the miR­206 precursor in cell­free (cf)RNA from the plasma of patients at risk of sarcopenia, and in cfRNAs from the muscles of mice subjected to muscle atrophy. Additionally, a decline in the levels of the miR­6516 precursor was observed in mice with muscle atrophy. The administration of mimic­miR­6516 to mice immobilized due to injury inhibited muscle atrophy by targeting and inhibiting cyclin­dependent kinase inhibitor 1b (Cdkn1b). Based on these results, the miR­206 precursor appears to be a potential biomarker of muscle atrophy, whereas miR­6516 shows promise as a therapeutic target to alleviate muscle deterioration in patients with muscle disuse and atrophy.

The Structural Adaptations That Mediate Disuse-Induced Atrophy of Skeletal Muscle.

The maintenance of skeletal muscle mass plays a fundamental role in health and issues associated with quality of life. Mechanical signals are one of the most potent regulators of muscle mass, with a decrease in mechanical loading leading to a decrease in muscle mass. This concept has been supported by a plethora of human- and animal-based studies over the past 100 years and has resulted in the commonly used term of 'disuse atrophy'. These same studies have also provided a great deal of insight into the structural adaptations that mediate disuse-induced atrophy. For instance, disuse results in radial atrophy of fascicles, and this is driven, at least in part, by radial atrophy of the muscle fibers. However, the ultrastructural adaptations that mediate these changes remain far from defined. Indeed, even the most basic questions, such as whether the radial atrophy of muscle fibers is driven by the radial atrophy of myofibrils and/or myofibril hypoplasia, have yet to be answered. In this review, we thoroughly summarize what is known about the macroscopic, microscopic, and ultrastructural adaptations that mediated disuse-induced atrophy and highlight some of the major gaps in knowledge that need to be filled.

A non-invasive mouse model that recapitulates disuse-induced muscle atrophy in immobilized patients.

Disuse muscle atrophy occurs consequent to prolonged limb immobility or bed rest, which represents an unmet medical need. As existing animal models of limb immobilization often cause skin erosion, edema, and other untoward effects, we here report an alternative method via thermoplastic immobilization of hindlimbs in mice. While significant decreases in the weight and fiber size were noted after 7 days of immobilization, no apparent skin erosion or edema was found. To shed light onto the molecular mechanism underlying this muscle wasting, we performed the next-generation sequencing analysis of gastrocnemius muscles from immobilized versus non-mobilized legs. Among a total of 55,487 genes analyzed, 787 genes were differentially expressed (> fourfold; 454 and 333 genes up- and down-regulated, respectively), which included genes associated with muscle tissue development, muscle system process, protein digestion and absorption, and inflammation-related signaling. From a clinical perspective, this model may help understand the molecular/cellular mechanism that drives muscle disuse and identify therapeutic strategies for this debilitating disease.

Plasticity changes in neuromuscular junction morphology and related regulatory proteins in the hibernating ground squirrel.

Skeletal muscle disuse atrophy can cause degenerative changes in neuromuscular junction morphology. Although Daurian ground squirrels (Spermophilus dauricus) are a natural anti-disuse animal model for studying muscle atrophy during hibernation, little is known about the morphological and regulatory mechanisms of their neuromuscular junctions. Here, we found that morphological indices of the soleus muscle were significantly lower during hibernation (torpor and interbout arousal) compared with pre-hibernation but recovered during post-hibernation. In the extensor digitorum longus muscle, neuromuscular junction morphology did not change significantly during hibernation. Agrin-Lrp4-MuSK is a key pathway for the formation and maintenance of the neuromuscular junction. Our results showed that low-density lipoprotein receptor-associated protein 4 (Lrp4) expression in the soleus (slow muscle) decreased by 46.2% in the interbout arousal group compared with the pre-hibernation group (P = 0.019), with recovery in the post-hibernation group. Compared with the pre-hibernation group, agrin expression in the extensor digitorum longus (fast muscle) increased by 67.0% in the interbout arousal group (P = 0.016). In conclusion, periodic up-regulation in agrin expression during interbout arousal may be involved in the maintenance of neuromuscular junction morphology in the extensor digitorum longus muscle during hibernation. The degenerative changes in neuromuscular junction morphology and the periodic decrease in Lrp4 protein expression in the soleus during hibernation, these changes recovered to the pre-hibernation levels in the post-hibernation group, exhibiting significant plasticity. This plasticity may be one of the important mechanisms for resisting disuse atrophy in hibernating animals.NEW & NOTEWORTHY This study is the first to explore the neuromuscular junction morphology of slow- and fast-twitch muscles in Daurian ground squirrels during different periods of hibernation. Results showed that the neuromuscular junction maintained stable morphology in the extensor digitorum longus muscle. The degenerative changes in neuromuscular junction morphology and the periodic decrease in Lrp4 protein expression in the soleus muscle during hibernation recovered in post-hibernation, exhibiting significant plasticity.

Global deletion of CCL2 has adverse impacts on recovery of skeletal muscle fiber size and function and is muscle specific.

Timely and complete recovery of muscle mass and function following a bout of physical disuse are critical components of returning to normal activities of daily living and lifestyle. Proper cross talk between the muscle tissue and myeloid cells (e.g., macrophages) throughout the recovery period from disuse atrophy plays a significant role in the complete resolution of muscle size and function. Chemokine C-C motif ligand 2 (CCL2) has a critical function of recruiting macrophages during the early phase of muscle damage. However, the importance of CCL2 has not been defined in the context of disuse and recovery. Here, we utilized a mouse model of whole body CCL2 deletion (CCL2KO) and subjected them to a period of hindlimb unloading followed by reloading to investigate the importance of CCL2 on the regrowth of muscle following disuse atrophy using ex vivo muscle tests, immunohistochemistry, and fluorescence-activated cell sorting approaches. We show mice that lack CCL2 display an incomplete recovery of gastrocnemius muscle mass, myofiber cross-sectional area, and EDL muscle contractile characteristics during the recovery from disuse atrophy. The soleus and plantaris had limited impact as a result of CCL2 deficiency suggesting a muscle-specific effect. Mice that lack CCL2 have decreased skeletal muscle collagen turnover, which may be related to defects in muscle function and stiffness. In addition, we show that the recruitment of macrophages to gastrocnemius muscle was dramatically reduced in CCL2KO mice during the recovery from disuse atrophy, which likely precipitated poor recovery of muscle size and function and aberrant collagen remodeling.NEW & NOTEWORTHY We provide evidence that the whole body loss of CCL2 in mice has adverse impacts on whole body function and skeletal muscle-specific contractile characteristics and collagen content. These defects in muscle function worsened during the recovery from disuse atrophy and corresponded with decreased recovery of muscle mass. We conclude that the absence of CCL2 decreased recruitment of proinflammatory macrophages to the muscle during the regrowth phase following disuse atrophy resulting in impaired collagen remodeling events and full resolution of muscle morphology and function.

Treadmill Exercise Suppresses Histological Progression of Disuse Atrophy in Articular Cartilage in Rat Knee Joints during Hindlimb Suspension.

OBJECTIVE: The purpose of this study was to determine the preventive effects of treadmill exercise or physiological loading on disuse atrophy in the rat knee joint cartilage and bone during hindlimb suspension. DESIGN: Twenty male rats were divided into 4 experimental groups, including the control, hindlimb suspension, physiological loading, and treadmill walking groups. Histological changes in the articular cartilage and bone of the tibia were histomorphometrically and immunohistochemically evaluated 4 weeks after the intervention. RESULTS: Compared with the control group, the hindlimb suspension group showed thinning of cartilage thickness, decreased matrix staining, and decreased proportion of noncalcified layers. Cartilage thinning, decreased matrix staining, and decreased noncalcified layers were suppressed in the treadmill walking group. The physiological loading group exhibited no significant suppression of cartilage thinning or decreased noncalcified layers, but the decreased matrix staining was significantly suppressed. No significant prevention of bone mass loss or changes in subchondral bone thickness were detected after physiological loading or treadmill walking. CONCLUSION: Disuse atrophy of the articular cartilage caused by unloading conditions could be prevented by treadmill walking in rat knee joints.

Loss of 4E-BPs prevents the hindlimb immobilization-induced decrease in protein synthesis in skeletal muscle.

The present study was designed to test the hypothesis that upregulating protein synthesis attenuates the loss of muscle mass in a model of disuse atrophy. The studies compared the effect of unilateral hindlimb immobilization in wild-type (WT) mice and double-knockout (DKO) mice lacking the translational regulators 4E-BP1 and 4E-BP2. Immobilization-induced downregulation of protein synthesis occurred in both groups of mice, but protein synthesis was higher in gastrocnemius muscle from the immobilized hindlimb of fasted DKO compared with WT mice. Surprisingly, although protein synthesis was partially elevated in DKO compared with WT mice, atrophy occurred to the same extent in both groups of animals. This may be partially due to impaired leucine-induced stimulation of protein synthesis in DKO compared with WT mice due to downregulated eukaryotic initiation factor eIF4E expression in muscle of DKO compared with WT mice. Expression of the E3 ubiquitin ligases MAFbx and MuRF-1 mRNAs and total protein ubiquitylation was upregulated in the immobilized compared with the nonimmobilized hindlimb of both WT and DKO mice, with little difference in the magnitude of the upregulation between genotypes. Analysis of newly synthesized proteins revealed downregulation of several glycolytic enzymes in the gastrocnemius of DKO mice compared with WT mice, as well as in the immobilized compared with the nonimmobilized hindlimb. Overall, the results suggest that the elevated rate of protein synthesis during hindlimb immobilization in fasted DKO mice is insufficient to prevent disuse-induced muscle atrophy, probably due to induction of compensatory mechanisms including downregulation of eIF4E expression.NEW & NOTEWORTHY Basal rates of protein synthesis are elevated in skeletal muscle in the immobilized leg of mice lacking the translational repressors, 4E-BP1 and 4E-BP2 (knockout mice), compared with wild-type mice. However, disuse-induced muscle atrophy occurs to the same extent in both wild-type and knockout mice suggesting that compensatory mechanisms are induced that overcome the upregulation of muscle protein synthesis. Proteomic analysis revealed that mRNAs encoding several glycolytic enzymes are differentially translated in wild-type and knockout mice.

Macrophage immunomodulation accelerates skeletal muscle functional recovery in aged mice following disuse atrophy.

Poor recovery of muscle size and strength with aging coincides with a dysregulated macrophage response during the early stages of regrowth. Immunomodulation in the form of ex vivo cytokine (macrophage-colony stimulating factor) or polarized macrophage delivery has been demonstrated to improve skeletal muscle regeneration. However, it is unclear if these macrophage-promoting approaches would be effective to improve skeletal muscle recovery following disuse in aged animals. Here, we isolated bone marrow-derived macrophages from donor mice of different ages under various experimental conditions and polarized them into proinflammatory macrophages. Macrophages were delivered intramuscularly into young adult or aged recipient mice during the early recovery period following a period of hindlimb unloading (HU). Delivery of proinflammatory macrophages from donor young adults or aged mice was sufficient to increase muscle function of aged mice during the recovery period. Moreover, proinflammatory macrophages derived from aged donor mice collected during recovery were similarly able to increase muscle function of aged mice following disuse. In addition to the delivery of macrophages, we showed that the intramuscular injection of the cytokine, macrophage-colony stimulating factor, to the muscle of aged mice following HU was able to increase muscle macrophage content and muscle force production during recovery. Together, these results suggest that macrophage immunomodulation approaches in the form of ex vivo proinflammatory macrophage or macrophage-colony stimulating factor delivery during the early recovery phase following disuse atrophy were sufficient to restore the loss of aged skeletal muscle function.NEW & NOTEWORTHY A single intramuscular administration of polarized macrophages into muscles of aged mice following a bout of disuse atrophy was sufficient to improve functional recover similarly to young adults after disuse atrophy regardless of the age or experimental condition of the donor mice. Additionally, intramuscular delivery of macrophage-colony stimulating factor into aged mice was similarly effective. Targeting macrophage function early during the regrowth phase may be a novel tool to bolster muscle recovery in aging.

Gomisin G improves muscle strength by enhancing mitochondrial biogenesis and function in disuse muscle atrophic mice.

Disuse muscle atrophy is characterized by a decrease in muscle mass and strength and an increase in glycolytic muscle fiber type. Although Schisandra chinensis extract has beneficial effects on muscle atrophy induced by various conditions (e.g., dexamethasone and aging), the effect of gomisin G, a lignan component of S. chinensis, on disuse muscle atrophy is unclear. Here, we induced disuse muscle atrophy through wire immobilization of the hind legs in mice followed by the oral administration of gomisin G. The cross-sectional area and muscle strength in disuse muscle atrophic mice were increased by gomisin G; however, the total muscle mass did not increase. Gomisin G decreased the expression of muscle atrophic factors (myostatin, atrogin-1, and MuRF1) but increased the expression of protein synthesis factors (mTOR and 4E-BP1). In H2O2-treated C2C12 myotubes, the level of puromycin incorporation (as a marker of protein synthesis) gradually increased in a dose-dependent manner by gomisin G. Furthermore, gomisin G induced a muscle fiber switch from fast-type glycolytic fibers (type 2B) to slow-type oxidative fibers (type I, 2A) in the gastrocnemius (GA) muscle as proved a decrease in the expression of TnI-FS and an increase in the expression of TnI-SS. Gomisin G increased mitochondrial DNA content and ATP levels in the GA muscle and COX activity in H2O2-treated C2C12 myotubes, improving mitochondrial function. Mechanistically, mitochondrial biogenesis is regulated by gomisin G via the Sirt 1/PGC-1α signaling pathway, targeting NRF1 and TFAM. These data suggest that gomisin G has a potential therapeutic effect on disuse muscle atrophy.

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.

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.

Physiological Reloading Recovers Histologically Disuse Atrophy of the Articular Cartilage and Bone by Hindlimb Suspension in Rat Knee Joint.

OBJECTIVE: This study aimed to clarify physiological reloading on disuse atrophy of the articular cartilage and bone in the rat knee using the hindlimb suspension model. DESIGN: Thirty male rats were divided into 3 experimental groups: control group, hindlimb suspension group, and reloading after hindlimb suspension group. Histological changes in the articular cartilage and bone of the tibia were evaluated by histomorphometrical and immunohistochemical analyses at 2 and 4 weeks after reloading. RESULTS: The thinning and loss of matrix staining in the articular cartilage and the decrease in bone volume induced by hindlimb suspension recovered to the same level as the control group after 2 weeks of reloading. The proportion of the noncalcified and calcified layers of the articular cartilage and the thinning of subchondral bone recovered to the same level as the control group after 4 weeks of reloading. CONCLUSIONS: Disuse atrophy of the articular cartilage and bone induced by hindlimb suspension in the tibia of rats was improved by physiological reloading.

Disuse muscle atrophy-improving effect of ninjin'yoeito in a mouse model.

Disuse syndrome indicates psychosomatic hypofunction caused by excess rest and motionless and muscle atrophy is termed disuse muscle atrophy. Disuse muscle atrophy-induced muscle weakness and hypoactivity further induces muscle atrophy, leading to a vicious cycle, and this is considered a factor causing secondary sarcopenia and subsequently frailty. Since frailty finally leads to a bedridden state requiring nursing, in facing a super-aging society, intervention for a risk factor of frailty, disuse muscle atrophy, is important. However, the main treatment of disuse muscle atrophy is physical therapy and there are fewer effective preventive and therapeutic drugs. The objective of this study was to search for Kampo medicine with a disuse muscle atrophy-improving effect. Ninjin'yoeito is classified as a qi-blood sohozai (dual supplement) in Chinese herbal medicine, and it has an action supplementing the spleen related to muscle. In addition, improvement of muscle mass and muscle weakness by ninjin'yoeito in a clinical study has been reported. In this study, the effect of ninjin'yoeito on disuse muscle atrophy was investigated. A disuse muscle atrophy model was prepared using male ICR mice. After surgery applying a ring for tail suspension, a 1-week recovery period was set. Ninjin'yoeito was administered by mixing it in the diet for 1 week after the recovery period, followed by tail suspension for 14 days. Ninjin'yoeito administration was continued until autopsy including the hindlimb suspension period. The mice were euthanized and autopsied immediately after completion of tail suspension, and the hindlimb muscles were collected. The food and water intakes during the hindlimb unloaded period, wet weight of the collected muscle, and muscle synthesis and muscle degradation-related factors in blood and muscle were evaluated. Ingestion of ninjin'yoeito inhibited tail suspension-induced reduction of the soleus muscle wet weight. In addition, an increase in the blood level of a muscle synthesis-related factor, IGF-1, and promotion of phosphorylation of mTOR and 4E-BP1 in the soleus muscle were observed. It was suggested that ninjin'yoeito has a disuse muscle atrophy-improving action. Promotion of the muscle synthesis pathway was considered the action mechanism of this.

The oestrous cycle and skeletal muscle atrophy: Investigations in rodent models of muscle loss.

NEW FINDINGS: What is the central question of this study? Is the oestrous cycle affected during disuse atrophies and, if so, how are oestrous cycle changes related to musculoskeletal outcomes? What is the main finding and its importance? Rodent oestrous cycles were altered during disuse atrophy, which was correlated with musculoskeletal outcomes. However, the oestrous cycle did not appear to be changed by Lewis lung carcinoma, which resulted in no differences in muscle size in comparison to healthy control animals. These findings suggest a relationship between the oestrous cycle and muscle size during atrophic pathologies. ABSTRACT: Recent efforts have focused on improving our understanding of female muscle physiology during exposure to muscle atrophic stimuli. A key feature of female rodent physiology is the oestrous cycle. However, it is not known how such stimuli interact with the oestrous cycle to influence muscle health. In this study, we investigated the impact of muscle atrophic stimuli on the oestrous cycle and how these alterations are correlated with musculoskeletal outcomes. A series of experiments were performed in female rodents, including hindlimb unloading (HU), HU followed by 24 h of reloading, HU combined with dexamethasone treatment, and Lewis lung carcinoma. The oestrous cycle phase was assessed throughout each intervention and correlated with musculoskeletal outcomes. Seven or 14 days of HU increased the duration in dioestrus or metoestrus (D/M; low hormones) and was negatively correlated with gastrocnemius mass. Time spent in D/M was also negatively correlated with changes in grip strength and bone density after HU, and with muscle recovery 24 h after the cessation of HU. The addition of dexamethasone strengthened these relationships between time in D/M and reduced musculoskeletal outcomes. However, in animals with Lewis lung carcinoma, oestrous cyclicity did not differ from that of control animals, and time spent in D/M was not correlated with either gastrocnemius mass or tumour burden. In vitro experiments suggested that enhanced protein synthesis induced by estrogen might protect against muscle atrophy. In conclusion, muscle atrophic insults are correlated with changes in the oestrous cycle, which are associated with deterioration in musculoskeletal outcomes. The magnitude of oestrous cycle alterations depends on the atrophic stimuli.

Disrupted macrophage metabolic reprogramming in aged soleus muscle during early recovery following disuse atrophy.

Aged skeletal muscle is characterized by poor muscle recovery following disuse coinciding with an impaired muscle pro-inflammatory macrophage response. Macrophage inflammatory status is regulated by its metabolic state, but little is understood of macrophage metabolism and its relation to macrophage inflammation in the context of muscle recovery and aging. Therefore, the purpose of this study was to thoroughly characterize macrophage metabolism and inflammation in aged muscle during early recovery following disuse atrophy using single cell transcriptomics and functional assays. Young (4-5 months) and old (20-22 months) male C57BL/6 mice underwent 14 days of hindlimb unloading followed by 4 days of ambulatory recovery. CD45+ cells were isolated from solei muscles and analyzed using 10x Genomics single cell RNA sequencing. We found that aged pro-inflammatory macrophage clusters were characterized with an impaired inflammatory and glycolytic transcriptome, and this dysregulation was accompanied by a suppression of HIF-1α and its immediate downstream target, Glut1. As a follow-up, bone marrow-derived macrophages were isolated from a separate cohort of young and old mice at 4-d recovery and were polarized to a pro-inflammatory phenotype and used for glycolysis stress test, phagocytosis activity assay, and targeted GC-MS metabolomics. Aged bone marrow-derived pro-inflammatory macrophages were characterized with impaired glycolysis and phagocytosis function, decreased succinate and an accumulation of glycolytic metabolic intermediates overall supporting reduced glycolytic flux and macrophage function. Our results indicate that the metabolic reprograming and function of aged skeletal muscle pro-inflammatory macrophages are dysfunctional during early recovery from disuse atrophy possibly attributing to attenuated regrowth.

The impact of hindlimb disuse on sepsis-induced myopathy in mice.

Sepsis induces a myopathy characterized by loss of muscle mass and weakness. Septic patients undergo prolonged periods of limb muscle disuse due to bed rest. The contribution of limb muscle disuse to the myopathy phenotype remains poorly described. To characterize sepsis-induced myopathy with hindlimb disuse, we combined the classic sepsis model via cecal ligation and puncture (CLP) with the disuse model of hindlimb suspension (HLS) in mice. Male C57bl/6j mice underwent CLP or SHAM surgeries. Four days after surgeries, mice underwent HLS or normal ambulation (NA) for 7 days. Soleus (SOL) and extensor digitorum longus (EDL) were dissected for in vitro muscle mechanics, morphological, and histological assessments. In SOL muscles, both CLP+NA and SHAM+HLS conditions elicited ~20% reduction in specific force (p < 0.05). When combined, CLP+HLS elicited ~35% decrease in specific force (p < 0.05). Loss of maximal specific force (~8%) was evident in EDL muscles only in CLP+HLS mice (p < 0.05). CLP+HLS reduced muscle fiber cross-sectional area (CSA) and mass in SOL (p < 0.05). In EDL muscles, CLP+HLS decreased absolute mass to a smaller extent (p < 0.05) with no changes in CSA. Immunohistochemistry revealed substantial myeloid cell infiltration (CD68+) in SOL, but not in EDL muscles, of CLP+HLS mice (p < 0.05). Combining CLP with HLS is a feasible model to study sepsis-induced myopathy in mice. Hindlimb disuse combined with sepsis induced muscle dysfunction and immune cell infiltration in a muscle dependent manner. These findings highlight the importance of rehabilitative interventions in septic hosts to prevent muscle disuse and help attenuate the myopathy.

Deletion of muscle Igf1 exacerbates disuse atrophy weakness in mice.

Muscle atrophy occurs as a result of prolonged periods of reduced mechanical stimulation associated with injury or disease. The growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and load sensing pathways can both aid in recovery from disuse through their shared downstream signaling, but their relative contributions to these processes are not fully understood. The goal of this study was to determine whether reduced muscle IGF-1 altered the response to disuse and reloading. Adult male mice with inducible muscle-specific IGF-1 deletion (MID) induced 1 wk before suspension and age-matched controls (CON) were subjected to hindlimb suspension and reloading. Analysis of muscle force, morphology, gene expression, signaling, and tissue weights was performed in nonsuspended (NS) mice, and those suspended for 7 days or reloaded following suspension for 3, 7, and 14 days. MID mice displayed diminished IGF-1 protein levels and muscle atrophy before suspension. Muscles from suspended CON mice displayed a similar extent of atrophy and depletion of IGF-1, yet combined loss of load and IGF-1 was not additive with respect to muscle mass. In contrast, soleus force generation capacity was diminished to the greatest extent when both suspension and IGF-1 deletion occurred. Recovery of mass, force, and gene expression patterns following suspension were similar in CON and MID mice, even though IGF-1 levels increased only in muscles from CON mice. Diminished strength in disuse atrophy is exacerbated with the loss of muscle IGF-1 production, whereas recovery of mass and strength upon reloading can occur even IGF-1 is low.NEW & NOTEWORTHY A mouse model with skeletal muscle-specific inducible deletion of Igf1 was used to address the importance of this growth factor for the consequences of disuse atrophy. Rapid and equivalent loss of IGF-I and mass occurred with deletion or disuse. Decrements in strength were most severe with combined loss of load and IGF-1. Return of mass and strength upon reloading was independent of IGF-1.

Age-Related Susceptibility to Muscle Damage Following Mechanotherapy in Rats Recovering From Disuse Atrophy.

The inability to fully recover lost muscle mass following periods of disuse atrophy predisposes older adults to lost independence and poor quality of life. We have previously shown that mechanotherapy at a moderate load (4.5 N) enhances muscle mass recovery following atrophy in adult, but not older adult rats. We propose that elevated transverse stiffness in aged muscle inhibits the growth response to mechanotherapy and hypothesize that a higher load (7.6 N) will overcome this resistance to mechanical stimuli. F344/BN adult and older adult male rats underwent 14 days of hindlimb suspension, followed by 7 days of recovery with (RE + M) or without (RE) mechanotherapy at 7.6 N on gastrocnemius muscle. The 7.6 N load was determined by measuring transverse passive stiffness and linearly scaling up from 4.5 N. No differences in protein turnover or mean fiber cross-sectional area were observed between RE and RE + M for older adult rats or adult rats at 7.6 N. However, there was a higher number of small muscle fibers present in older adult, but not adult rats, which was explained by a 16-fold increase in the frequency of small fibers expressing embryonic myosin heavy chain. Elevated central nucleation, satellite cell abundance, and dystrophin-/laminin+ fibers were present in older adult rats only following 7.6 N, while 4.5 N did not induce damage at either age. We conclude that age is an important variable when considering load used during mechanotherapy and age-related transverse stiffness may predispose older adults to damage during the recovery period following disuse atrophy.

Chronic Ouabain Prevents Na,K-ATPase Dysfunction and Targets AMPK and IL-6 in Disused Rat Soleus Muscle.

Sustained sarcolemma depolarization due to loss of the Na,K-ATPase function is characteristic for skeletal muscle motor dysfunction. Ouabain, a specific ligand of the Na,K-ATPase, has a circulating endogenous analogue. We hypothesized that the Na,K-ATPase targeted by the elevated level of circulating ouabain modulates skeletal muscle electrogenesis and prevents its disuse-induced disturbances. Isolated soleus muscles from rats intraperitoneally injected with ouabain alone or subsequently exposed to muscle disuse by 6-h hindlimb suspension (HS) were studied. Conventional electrophysiology, Western blotting, and confocal microscopy with cytochemistry were used. Acutely applied 10 nM ouabain hyperpolarized the membrane. However, a single injection of ouabain (1 µg/kg) prior HS was unable to prevent the HS-induced membrane depolarization. Chronic administration of ouabain for four days did not change the α1 and α2 Na,K-ATPase protein content, however it partially prevented the HS-induced loss of the Na,K-ATPase electrogenic activity and sarcolemma depolarization. These changes were associated with increased phosphorylation levels of AMP-activated protein kinase (AMPK), its substrate acetyl-CoA carboxylase and p70 protein, accompanied with increased mRNA expression of interleikin-6 (IL-6) and IL-6 receptor. Considering the role of AMPK in regulation of the Na,K-ATPase, we suggest an IL-6/AMPK contribution to prevent the effects of chronic ouabain under skeletal muscle disuse.