Hindlimb Immobilization Research Articles

Ameliorative Effects of Fermented Red Ginseng Extract on Muscle Atrophy in Dexamethasone-Induced C2C12 Cell And Hind Limb-Immobilized C57BL/6J Mice.

Fermented red ginseng (FRG) enhances the bioactivity and bioavailability of ginsenosides, which possess various immunomodulatory, antiaging, anti-obesity, and antidiabetic properties. However, the effects of FRG extract on muscle atrophy and the underlying molecular mechanisms remain unclear. This study aimed to elucidate the effects of FRG extract on muscle atrophy using both in vitro and invivo models. In vitro experiments used dexamethasone (DEX)-induced C2C12 myotubes to assess cell viability, myotube diameter, and fusion index. In vivo experiments were conducted on hind limb immobilization (HI)-induced mice to evaluate grip strength, muscle mass, and fiber cross-sectional area (CSA) of the gastrocnemius (GAS), quadriceps (QUA), and soleus (SOL) muscles. Molecular mechanisms were investigated through the analysis of key signaling pathways associated with muscle protein synthesis, energy metabolism, and protein degradation. FRG extract treatment enhanced viability of DEX-induced C2C12 myotubes and restored myotube diameter and fusion index. In HI-induced mice, FRG extract improved grip strength, increased muscle mass and CSA of GAS, QUA, and SOL muscles. Mechanistic studies revealed that FRG extract activated the insulin-like growth factor 1/protein kinase B (Akt)/mammalian target of rapamycin signaling pathway, promoted muscle energy metabolism via the sirtuin 1/peroxisome proliferator-activated receptor gamma-coactivator-1α pathway, and inhibited muscle protein degradation by suppressing the forkhead box O3a, muscle ring-finger 1, and F-box protein (Fbx32) signaling pathways. FRG extract shows promise for ameliorating muscle atrophy by modulating key molecular pathways associated with muscle protein synthesis, energy metabolism, and protein degradation, offering insights for future drug development.

Hindlimb immobilization induces insulin resistance and elevates mitochondrial ROS production in the hippocampus of female rats.

Alzheimer's disease (AD) is the fifth leading cause of death in older adults, and treatment options are severely lacking. Recent findings demonstrate a strong relationship between skeletal muscle and cognitive function, with evidence supporting that muscle quality and cognitive function are positively correlated in older adults. Conversely, decreased muscle function is associated with a threefold increased risk of cognitive decline. Based on these observations, the purpose of this study was to investigate the negative effects of muscle disuse [via a model of hindlimb immobilization (HLI)] on hippocampal insulin sensitivity and mitochondrial function and identify the potential mechanisms involved. HLI for 10 days in 4-mo-old female Wistar rats resulted in the following novel findings: 1) hippocampal insulin resistance and deficits in whole body glucose homeostasis, 2) dramatically increased mitochondrial reactive oxygen species (ROS) production in the hippocampus, 3) elevated markers for amyloidogenic cleavage of amyloid precursor protein (APP) and tau protein in the hippocampus, 4) and reduced brain-derived neurotrophic factor (BDNF) expression. These findings were associated with global changes in iron homeostasis, with muscle disuse producing muscle iron accumulation in association with decreased serum and whole brain iron levels. We report the novel finding that muscle disuse alters brain iron homeostasis and reveal a strong negative correlation between muscle and brain iron content. Overall, HLI-induced muscle disuse has robust negative effects on hippocampal insulin sensitivity and ROS production in association with altered brain iron homeostasis. This work provides potential novel mechanisms that may help explain how loss of muscle function contributes to cognitive decline and AD risk.NEW & NOTEWORTHY Muscle disuse via hindlimb immobilization increased oxidative stress and insulin resistance in the hippocampus. These findings were in association with muscle iron overload in connection with iron dysregulation in the brain. Overall, our work identifies muscle disuse as a contributor to hippocampal dysfunction, potentially through an iron-based muscle-brain axis, highlighting iron dysregulation as a potential novel mechanism in the relationship between muscle health, cognitive function, and Alzheimer's disease risk.

Infrared thermography and computed tomography imaging for hind limb study after immobilization-induced disuse atrophy

Immobilization for treatment after an injury can lead to disuse atrophy, resulting in reduced functionality and strength of the immobilized limb. In our study, we utilized infrared thermography (IR) and computed tomography (CT) ex vivo to assess both physiological and structural changes following hind limb immobilization in a young Wistar rat model. Twelve rats weighing 275 ± 30 g had their right hind limbs immobilized with a modified Thomas-splint for varying durations (3, 7, or 14 days). IR imaging using an infrared camera provided insight into limb temperature changes. For micro-CT, we implemented a stain-ethanol fixation method and a gray score which enabled us to visualize and quantify muscle alterations. Thermographic images showed an increase in temperature of up to 8% in the hind limb at supine position at 14 days due to the inflammatory process while micro-CT exhibited muscle shrinkage of 10 and 18% at 7 and 14 days, respectively. Our findings underscore the efficacy of IR and micro-CT as rapid and precise imaging modalities for detecting morphological shifts in muscle tissue, particularly in pathological conditions like atrophy.

Therapeutic Effect of AAV8-Mediated miR-23a in Immobilization-Induced Muscle Atrophy in Mice

bjective: To investigate the therapeutic effect of miR-23a in immobilization-induced muscle atrophy in mice. Methods: (Experiment 1) Twelve C57BL6 wild-type mice were randomly divided into two groups: Sham group (sham surgery) and Immobilization (hind limb immobilization surgery). Fluorescence quantitative PCR was performed to detect the expression changes of miR-23a, MuRF-1, and Atrogin-1 7 days post-surgery. (Experiment 2) Twelve C57BL6 wild-type mice were randomly divided into 4 groups according to surgery type (sham surgery and hind limb immobilization surgery) and injection type (AAV8 or AAV8-OEmiR-23a), fluorescence quantitative PCR to detect changes in MuRF-1 and Atrogin-1 expression in mouse gastrocnemius muscle. (Experiment 3) NFATc3 expression changes were detected by fluorescence quantitative PCR 48 hours post-transfection. (Experiment 4) Twelve C57BL6 wild-type mice underwent hind limb immobilization surgery, divided into 4 groups : Immobilization+AAV8-control, Immobilization+AAV8-OEmiR23a, Immobilization+AAV8-shNFATc3, and Immobilization+AAV8-OEmiR-23a+AAV8-shNFATc3. quantitative PCR to detect changes in MuRF-1 and Atrogin-1 expression in mouse gastrocnemius muscle. Results: Fluorescence quantitative PCR results showed that miR-23a was downregulated in immobilization-induced muscle atrophy, while MuRF-1 and Atrogin-1 expression was upregulated. WGA staining results showed that intramuscular injection of AAV8-OEmiR-23a could significantly alleviate the decrease in muscle fiber cross-sectional area and the increase in expression of muscle atrophy marker genes MuRF-1 and Atrogin-1 caused by hind limb immobilization, Fluorescence quantitative PCR revealed that when miR-23a expression was inhibited, NFATc3 expression was downregulated; when miR-23a was overexpressed, NFATc3 expression was upregulated, Fluorescence quantitative PCR results showed that intramuscular injection of AAV8-OEmiR-23a and AAV8-shNFATc3 could not alleviate the increase in expression of muscle atrophy marker genes MuRF-1 and Atrogin-1 caused by hind limb immobilization. Conclusion: miR-23a can treat hind limb immobilization-induced muscle atrophy in mice.

Combined orchiectomy and limb immobilization recapitulate early age‐related changes to skeletal muscle in mice

AbstractBackgroundMuscle mass and function decline in middle age, ultimately resulting in sarcopenia in the elderly and poor health outcomes, reducing quality of life. There is a lack of cost‐ and time‐effective murine models that recapitulate the physiological changes associated with muscle mass decline to study possible interventions to delay sarcopenia. We aimed to evaluate the effectiveness of combining orchiectomy (ORC) surgery to simulate age‐related androgen decline and hindlimb immobilization (IM) in inducing age‐related skeletal muscle changes.MethodsFour‐month‐old male C57BL/6J mice (n = 10) were subjected to ORC, followed by IM (right hindlimb casting) for 14 days. Upon completion of the casting period, ex vivo muscle contractile function, histology, and various mitochondrial markers were assessed, and results were compared with age‐matched controls (CON; n = 8) and middle‐aged (MA; 12 ± 1 months, n = 9) animals.ResultsIM combined with ORC induced a 30%–40% decrease in muscle mass across multiple hindlimb muscles (P < 0.0001), with the magnitude of muscle loss comparable with the MA group when corrected for body weight (P < 0.0001). In the IM limb of ORC mice, soleus muscle force significantly decreased when compared with the contralateral limb (P < 0.05) and aged‐matched CON group (P < 0.05). The decrements in muscle force and mass present in the IM limb of ORC mice were accompanied by a 70% reduction in the expression of the muscle structural protein dystrophin and various mitochondrial markers, including cytochrome C (−55%), peroxisome proliferator‐activated receptor gamma co‐activator 1‐beta (PGC1‐β) (−49%), and cytochrome oxidase IV (COX‐IV) (−73%) when compared with CON animals (P < 0.001). Lastly, our model also demonstrated specific fibre‐type shifts in fast‐ and slow‐twitch muscles, which mimicked changes in the MA group.ConclusionsApplying treatments during IM could target acute muscle atrophy in MA adults, while applying them following cast removal in a low‐testosterone environment could represent a window for rehabilitation therapeutics.

DBC1maintains skeletal muscle integrity by enhancing myogenesis and preventing myofibre wasting.

Skeletal muscle atrophy, particularly ageing-related muscular atrophy such as sarcopenia, is a significant health concern. Despite its prevalence, the underlying mechanisms remain poorly understood, and specific approved medications are currently unavailable. Deleted in breast cancer 1 (DBC1) is a well-known regulator of senescence, metabolism or apoptosis. Recent reports suggest that DBC1 may also potentially regulate muscle function, as mice lacking DBC1 exhibit weakness and limpness. However, the function of DBC1 in skeletal muscle and its associated molecular mechanisms remain unknown, thus prompting the focus of this study. Tibialis anterior (TA) muscle-specific DBC1 knockdown C57BL/6J male mice were generated through a single injection of 2.00 E+11vg of adeno-associated virus 9 delivering single-guide RNA for DBC1. Grip strength and endurance were assessed 2months later, followed by skeletal muscle harvest. Muscle atrophy model was generated by cast immobilization of the mouse hindlimb for 2weeks. Molecular markers of atrophy were probed in muscles upon termination. Cardiotoxin (CTX) was injected in TA muscles of DBC1 knockdown mice, and muscle regeneration was assessed by immunohistochemistry, quantitative PCR and western blotting. DBC1 knockdown C2C12 cells and myotubes were investigated using immunofluorescence staining, Seahorse, immunohistology, fluorescence-activated cell sorting and RNA-sequencing analyses. DBC1 knockdown in skeletal muscle of young mice led to signatures of muscle atrophy, including a 28% reduction in muscle grip force (P=0.023), a 54.4% reduction in running distance (P=0.002), a 14.3% reduction in muscle mass (P=0.007) and significantly smaller myofibre cross-sectional areas (P<0.0001). DBC1 levels decrease in age-related or limb immobilization-induced atrophic mouse muscles and overexpress DBC1-attenuated atrophic phenotypes in these mice. Muscle regeneration was hampered in mice with CTX-induced muscle injury by DBC1 knockdown, as evidenced by reductions in myofibre cross-sectional areas of regenerating myofibres with centralized nuclei (P<0.0001), percentages of MyoG+ nuclei (P<0.0001) and fusion index (P<0.0001). DBC1 transcriptionally regulated mouse double minute 2 (MDM2), which mediated ubiquitination and degradation of forkhead box O3 (FOXO3). Increased FOXO3 proteins hampered myogenesis in DBC1 knockdown satellite cells by compromising around 50% of mitochondrial functions (P<0.001) and exacerbated atrophy in DBC1 knockdown myofibres by activating the ubiquitin-proteasome and autophagy-lysosome pathways. DBC1 is essential in maintaining skeletal muscle integrity by protecting against myofibres wasting and enhancing muscle regeneration via FOXO3. This research highlights the significance of DBC1 for healthy skeletal muscle function and its connection to muscular atrophy.

Abstract 18258: Leucine Supplementation Improves Myocardial Function in a HFpEF Rat Model

Introduction: We previously documented that leucine supplementation protects from skeletal muscle atrophy by increasing skeletal muscle mass and force during hind limb immobilization. One of the proposed underlying mechanisms was the suppression of HDAC4 protein expression and increased nuclei accretion. Hypothesis: The present study aimed to examine whether the protective effects of leucine supplementation are also observed in the myocardium in an animal model of heart failure with preserved ejection fraction (HFpEF) and to assess the role of HDAC4 modulation. Methods: Female ZSF1 lean (control, con) and obese (HFpEF) rats were randomized at an age of 20 weeks to one of three groups: lean rats receiving standard chow (con), obese rats receiving standard chow (HFpEF), and obese rats receiving leucine-supplemented chow (3% w/w) for 12 weeks (HFpEF/leu). All animals underwent echocardiography at baseline and after 6 and 12 weeks. Prior to organ harvest left ventricular (LV) hemodynamics were measured invasively, blood samples were taken, cardiac mitochondrial function was assessed and LV tissue was collected for molecular and histological analyses. Results: Assessment of diastolic function revealed a 20% reduction of E/é in the HFpEF/leu group compared to untreated HFpEF. While no alterations of left ventricular hemodynamics were observed between the groups, stiffness factor β was significantly reduced in leucine-treated rats. Left ventricular mRNA levels of Collagen type I a1 and matrix metalloproteinase 2 were reduced by 25% and 22%, respectively. Assessment of mitochondrial oxidative phosphorylation revealed a significant improvement of complex-I- and complex-II mediated respiration in leucine-treated rats compared to HFpEF. Finally, HDAC4 protein expression levels were reduced by 40% in the HFpEF/leu group compared to HFpEF. Conclusions: This study presents beneficial effects of leucine supplementation on cardiac function, remodeling, and metabolic parameters in an HFpEF rat model. These benefits may be attributed to suppressed HDAC4 expression.

Therapeutic Potential of Hydrogen-Rich Water on Muscle Atrophy Caused by Immobilization in a Mouse Model.

Skeletal muscle atrophy is associated with poor quality of life and disability. Thus, finding a new strategy for the prevention and treatment of skeletal muscle atrophy is very crucial. This study aimed to investigate the therapeutic potential of hydrogen-rich water (HRW) on muscle atrophy in a unilateral hind limb immobilization model. Thirty-six male Balb/C mice were divided into control (without immobilization), atrophy, and atrophy + hydrogen-rich water (HRW). Unilateral hind limb immobilization was induced using a splint for 7 days (atrophy) and removed for 10 days (recovery). At the end of each phase, gastrocnemius and soleus muscle weight, limb grip strength, skeletal muscle histopathology, muscle fiber size, cross-section area (CSA), serum troponin I and skeletal muscle IL-6, TNF-α and Malondialdehyde (MDA), and mRNA expression of NF-κB, BAX and Beclin-1 were evaluated. Muscle weight and limb grip strength in the H2-treated group were significantly improved during the atrophy phase, and this improvement continued during the recovery period. Treatment by HRW increased CSA and muscle fiber size and reduced muscle fibrosis, serum troponin I, IL-6, TNF-α and MDA which was more prominent in the atrophy phase. These data suggest that HRW could improve muscle atrophy in an immobilized condition and could be considered a new strategy during rehabilitation.

Poster 120: Understanding the Biological Basis of Contracture Using a Mouse Model of Single Hindlimb Immobilization-Induced Fibrosis

Poster 120: Understanding the Biological Basis of Contracture Using a Mouse Model of Single Hindlimb Immobilization-Induced Fibrosis

승산(承山)경혈 자극이 흰쥐의 불용성 근위축에 미치는 효과

Skeletal muscle is the largest organ in the human body, comprising about 40% of the body weight. The prolonged periods of skeletal muscle inactivity due to bed rest, denervation, hindlimb unloading, or immobilization can result in significant muscle atrophy. It is characterized by decreased muscle fiber cross-sectional area and altered proteins. Apoptosis is known to contribute to disuse-induced skeletal muscle atrophy. Chengshan(BL57) on the lower leg is one of the most frequently used point in acupressure and acupuncture. In this study, the hypothesis that the acupressure at Chengshan could attenuate immobilization-induced skeletal muscle atrophy was tested.BR Thirty male Sprague Dawley Rats were divided into 3 groups: Normal(n=10), Control(n=10). AP (acupressure)(n=10). The left hindlimb immobilization was performed with casting tape in both Control group and AP group. The Rats in AP group were daily treated with pressure needle at the accupoint of Chengshan. After 2 weeks, the weight, cross sectional aera of left soleus muscle were assessed. The changes of apoptosis related proteins were observed.BR In results, AP group significantly ameliorated the reductions of the muscle weight and cross sectional area induced by immobilization. Moreover, BAX immunoreactivities was decreased and Bcl-2 was increased as compared with Control group. Theses results suggest that acupressure at Chengshan has the protective effects against disuse muscle atrophy by modulating BAX/Bcl-2 proteins in soleus muscle.

Mesenchymal stromal cells ameliorate diabetes-induced muscle atrophy through exosomes by enhancing AMPK/ULK1-mediated autophagy.

Diabetes and obesity are associated with muscle atrophy that reduces life quality and lacks effective treatment. Mesenchymal stromal cell (MSC)-based therapy can ameliorate high fat-diet (HFD) and immobilization (IM)-induced muscle atrophy in mice. However, the effect of MSCs on muscle atrophy in type 2 diabetes mellitus (T2DM) and the potential mechanism is unclear. Here, we evaluated the efficacy and explored molecular mechanisms of human umbilical cord MSCs (hucMSCs) and hucMSC-derived exosomes (MSC-EXO) on diabetes- and obesity-induced muscle atrophy. Diabetic db/db mice, mice fed with high-fat diet (HFD), mice with hindlimb immobilization (IM), and C2C12 myotubes were used to explore the effect of hucMSCs or MSC-EXO in alleviating muscle atrophy. Grip strength test and treadmill running were used to measure skeletal muscle strength and performance. Body composition, muscle weight, and muscle fibre cross-sectional area (CSA) was used to evaluate muscle mass. RNA-seq analysis of tibialis anterior (TA) muscle and Western blot analysis of muscle atrophy signalling, including MuRF1 and Atrogin 1, were performed to investigate the underlying mechanisms. hucMSCs increased grip strength (P=0.0256 in db/db mice, P=0.012 in HFD mice, P=0.0097 in IM mice), running endurance (P=0.0154 in HFD mice, P=0.0006 in IM mice), and muscle mass (P=0.0004 in db/db mice, P=0.0076 in HFD mice, P=0.0144 in IM mice) in all models tested, with elevated CSA of muscle fibres (P<0.0001 in db/db mice and HFD mice, P=0.0088 in IM mice) and reduced Atrogin1 (P=0.0459 in db/db mice, P=0.0088 in HFD mice, P=0.0016 in IM mice) and MuRF1 expression (P=0.0004 in db/db mice, P=0.0077 in HFD mice, P=0.0451 in IM mice). MSC-EXO replicated all these hucMSC-mediated changes (P=0.0103 for grip strength, P=0.013 for muscle mass, P<0.0001 for CSA of muscle fibres, P=0.0171 for Atrogin1 expression, and P=0.006 for MuRF1 expression). RNA-seq revealed that hucMSCs activated the AMPK/ULK1 signalling and enhanced autophagy. Knockdown of AMPK or inhibition of autophagy with 3-methyladenine (3-MA) diminished the beneficial anti-atrophy effects of hucMSCs or MSC-EXO. Our results suggest that human umbilical cord mesenchymal stromal cells mitigate diabetes- and obesity-induced muscle atrophy via enhancing AMPK/ULK1-mediated autophagy through exosomes, with implications of applying hucMSCs or hucMSC-derived exosomes to treat muscle atrophy.

Acupotomy relieves pain and improves motor function by regulating autophagy and apoptosis of cartilage in rabbits with knee osteoarthritis apoptosis

To observe the effect of acupotomy on the expression of Beclin-1, Bcl-2 and Caspase-3 in the cartilage tissue in rabbits with knee osteoarthritis (KOA), so as to explore its mechanism underling improvement of KOA. Twenty-four healthy male New Zealand rabbits were randomly and equally divided into blank control, model and acupotomy groups, with 8 rabbits in each group. By using the modified Videman's methods, the KOA model was established by left hind limb immobilization with a plaster cast for 6 weeks. The severity of KOA (knee pain, swelling and motor function) was assessed using Lequesne score, and the rabbits with a score below 4 were excluded. The acupotomy was applied to "Hedingci" (the attachment of the quadriceps tendon to the patella at the upper edge), "Binneixia" (the medial patellar supporting band attachment of medial inferior patellar margin), "Binwaixia" (the lateral patellar supporting band attachment of the lower lateral patellar margin), "Chengfeijian" (the lateral collateral ligament of the knee passes over the lateral joint space), "Weiyangci" (the medial margin of biceps femoris at the lateral end of popliteus), "Yinlingci" (the medial tibial attachment of anserinus tendon) on the left hind limb once a week for 4 weeks. One week after the last intervention, the left knee joint dysfunction severity(pain, maximum walking distance, and some activities of daily living) was evaluated by using modified Lequesne score. Histopathological changes of the cartilage were observed under light microscope after H.E. staining. The apoptosis of chondrocytes was observed after terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labeling (TUNEL) staining. The autophagolysosomes of chondrocytes were observed using transmission electron microscopy. The expression levels of Beclin-1, Bcl-2 and Caspase-3 (related factors of autophagy and apoptosis) were detected using Real-time PCR and Western blot separately. In comparison with the blank control group, the Lequesne score, apoptosis rate, expression levels of Caspase-3 mRNA and protein were significantly increased (P<0.001), and the number of autophagolysosomes, expression levels of Beclin-1 and Bcl-2 mRNAs and proteins considerably decreased (P<0.001) in the model group. Relevant to the model group, the acupotomy group had an obvious decrease in Lequesne score, rate of apoptosis, and expression levels of Caspase-3 mRNA and protein (P<0.001) and an apparent increase in the number of autophagolysosomes and expression levels of Beclin-1 and Bcl-2 mRNAs and proteins (P<0.001). Findings of H.E. staining showed severe damaged cartilage surface, with a large number of exfoliation defects, few chondrocytes on the surface and disordered arrangement of transitional cells in the model group, which was relatively milder in the acupotomy group. Acupotomy can mitigate knee-joint pain and improve functional activity in KOA rabbits, which may be associated with its functions in promoting autophagy and suppressing apoptosis by up-regulating expressions of Beclin-1 and Bcl-2 mRNAs and proteins and down-regulation of Caspase-3 mRNA and protein.

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.

4E‐BP1/2 Deletion Enhances mRNA Cap‐binding Complex Assembly and Protein Synthesis in Immobilized Skeletal Muscle But is Not Sufficient to Prevent Muscle Atrophy

Skeletal muscle atrophy in long‐term care patients is positively correlated with worse health outcomes and patient mortality. Immobilization‐induced muscle atrophy is due to an imbalance between protein synthesis and protein degradation. The imbalance favors degradation due to anabolic resistance within the muscle. Targeting regulators of protein synthesis to increase mRNA translation under immobilized conditions may improve the protein synthesis/degradation imbalance and ameliorate immobilization‐induced skeletal muscle atrophy. 4E‐BP1 and 4E‐BP2 (eukaryotic initiation factor 4E (eIF4E) binding proteins 1 and 2) are important regulators of mRNA translation initiation that bind to eIF4E and prevents its binding to the scaffold protein eIF4G, an essential and rate‐limiting step of cap‐dependent mRNA translation. Previous studies have implicated eIF4E and its binding proteins in disuse‐induced repression of muscle protein synthesis. Consequently, we hypothesize that eliminating 4E‐BP1/2 may enhance mRNA translation and protect against disuse‐induced muscle atrophy.The hindlimb muscles of 8–9‐week‐old C57Bl/6 mixed‐sex wild‐type (WT) (20.25±2.39 g; n=19) and global 4E‐BP1/4E‐BP2 double knockout (DKO) (20.55±2.65 g; n=19) mice were compared after 3 days of unilateral hindlimb immobilization. Each mouse had one hindlimb immobilized in the plantarflexed position while the contralateral nonimmobilized hindlimb served as a control. Food and water were provided to the mice ad libitum. The hindlimb muscles were removed after a 4‐6 h fast, and the weights were compared between both genotypes for both the immobilized and free limbs. The gastrocnemius muscles were homogenized, eIF4E was immunoprecipitated, and eIF4E, eIF4G, and 4E‐BP1 in the immunoprecipitated sample was assessed by western blot analysis. In the immobilized gastrocnemius of the DKO mice the amount of eIF4G associated with eIF4E was higher than in control mice (p=0.001). In addition, mRNA translation, as assessed in a separate study by the incorporation of puromycin (0.04 μmol puromycin/g of body mass) into protein in the gastrocnemius, was also higher in muscle of the immobilized leg of DKO (n=24) compared to control mice (n=26) (17.1% increase, p=0.012). Surprisingly, eIF4E binding protein deficiency did not prevent muscle atrophy, as the mass of the gastrocnemius, plantaris, and soleus muscles was reduced to a similar extent in the immobilized compared to the control leg of both DKO and wildtype mice. To assess a possible role for upregulation of protein degradation in disuse‐induced muscle atrophy, expression of the E3 ubiquitin ligase, MAFbx‐1 (muscle atrophy f‐box 1) was also assessed. MAFbx‐1 expression was elevated in the gastrocnemius of the immobilized compared to the contralateral non‐immobilized limb, though no significant difference was found between genotypes. More research into other components of the mRNA cap binding complex, e.g., eIF4A, and additional biomarkers of protein degradative pathways may reveal more details on the mechanism of immobilization‐induced muscle atrophy.

Effect of short-term hindlimb immobilization on skeletal muscle atrophy and the transcriptome in a low compared with high responder to endurance training model.

Skeletal muscle atrophy is a physiological response to disuse, aging, and disease. We compared changes in muscle mass and the transcriptome profile after short-term immobilization in a divergent model of high and low responders to endurance training to identify biological processes associated with the early atrophy response. Female rats selectively bred for high response to endurance training (HRT) and low response to endurance training (LRT; n = 6/group; generation 19) underwent 3 day hindlimb cast immobilization to compare atrophy of plantaris and soleus muscles with line-matched controls (n = 6/group). RNA sequencing was utilized to identify Gene Ontology Biological Processes with differential gene set enrichment. Aerobic training performed prior to the intervention showed HRT improved running distance (+60.6 ± 29.6%), while LRT were unchanged (-0.3 ± 13.3%). Soleus atrophy was greater in LRT vs. HRT (-9.0 ±8.8 vs. 6.2 ±8.2%; P<0.05) and there was a similar trend in plantaris (-16.4 ±5.6% vs. -8.5 ±7.4%; P = 0.064). A total of 140 and 118 biological processes were differentially enriched in plantaris and soleus muscles, respectively. Soleus muscle exhibited divergent LRT and HRT responses in processes including autophagy and immune response. In plantaris, processes associated with protein ubiquitination, as well as the atrogenes (Trim63 and Fbxo32), were more positively enriched in LRT. Overall, LRT demonstrate exacerbated atrophy compared to HRT, associated with differential gene enrichments of biological processes. This indicates that genetic factors that result in divergent adaptations to endurance exercise, may also regulate biological processes associated with short-term muscle unloading.

Hindlimb Immobilization Increases IL-1β and Cdkn2a Expression in Skeletal Muscle Fibro-Adipogenic Progenitor Cells: A Link Between Senescence and Muscle Disuse Atrophy.

Loss of muscle mass and strength contributes to decreased independence and an increased risk for morbidity and mortality. A better understanding of the cellular and molecular mechanisms underlying muscle atrophy therefore has significant clinical and therapeutic implications. Fibro-adipogenic progenitors (FAPs) are a skeletal muscle resident stem cell population that have recently been shown to play vital roles in muscle regeneration and muscle hypertrophy; however, the role that these cells play in muscle disuse atrophy is not well understood. We investigated the role of FAPs in disuse atrophy in vivo utilizing a 2-week single hindlimb immobilization model. RNA-seq was performed on FAPs isolated from the immobilized and non-immobilized limb. The RNAseq data show that IL-1β is significantly upregulated in FAPs following 2 weeks of immobilization, which we confirmed using droplet-digital PCR (ddPCR). We further validated the RNA-seq and ddPCR data from muscle in situ using RNAscope technology. IL-1β is recognized as a key component of the senescence-associated secretory phenotype, or SASP. We then tested the hypothesis that FAPs from the immobilized limb would show elevated senescence measured by cyclin-dependent kinase inhibitor 2A (Cdkn2a) expression as a senescence marker. The ddPCR and RNAscope data both revealed increased Cdkn2a expression in FAPs with immobilization. These data suggest that the gene expression profile of FAPs is significantly altered with disuse, and that disuse itself may drive senescence in FAPs further contributing to muscle atrophy.

Early movement restriction deteriorates motor function and soleus muscle physiology

Children with low physical activity and interactions with environment experience atypical sensorimotor development and maturation leading to anatomical and functional disorganization of the sensorimotor circuitry and also to enduring altered motor function. Previous data have shown that postnatal movement restriction in rats results in locomotor disturbances, functional disorganization and hyperexcitability of the hind limb representations in the somatosensory and motor cortices, without apparent brain damage. Due to the reciprocal interplay between the nervous system and muscle, it is difficult to determine whether muscle alteration is the cause or the result of the altered sensorimotor behavior (Canu et al., 2019). In the present paper, our objectives were to evaluate the impact of early movement restriction leading to sensorimotor restriction (SMR) during development on the postural soleus muscle and on sensorimotor performance in rats, and to determine whether changes were reversed when typical activity was resumed. Rats were submitted to SMR by hind limb immobilization for 16 h / day from birth to postnatal day 28 (PND28). In situ isometric contractile properties of soleus muscle, fiber cross sectional area (CSA) and myosin heavy chain content (MHC) were studied at PND28 and PND60. In addition, the motor function was evaluated weekly from PND28 to PND60. At PND28, SMR rats presented a severe atrophy of soleus muscle, a decrease in CSA and a force loss. The muscle maturation appeared delayed, with persistence of neonatal forms of MHC. Changes in kinetic properties were moderate or absent. The Hoffmann reflex provided evidence for spinal hyperreflexia and signs of spasticity. Most changes were reversed at PND60, except muscle atrophy. Functional motor tests that require a good limb coordination, i.e. rotarod and locomotion, showed an enduring alteration related to SMR, even after one month of ‘typical’ activity. On the other hand, paw withdrawal test and grip test were poorly affected by SMR whereas spontaneous locomotor activity increased over time. Our results support the idea that proprioceptive feedback is at least as important as the amount of motor activity to promote a typical development of motor function. A better knowledge of the interplay between hypoactivity, muscle properties and central motor commands may offer therapeutic perspectives for children suffering from neurodevelopmental disorders.

Beneficial Effects of Resveratrol in Mouse Gastrocnemius: A Hint to Muscle Phenotype and Proteolysis.

We hypothesized that the phenolic compound resveratrol mitigates muscle protein degradation and loss and improves muscle fiber cross-sectional area (CSA) in gastrocnemius of mice exposed to unloading (7dI). In gastrocnemius of mice (female C57BL/6J, 10 weeks) exposed to a seven-day period of hindlimb immobilization with/without resveratrol treatment, markers of muscle proteolysis (tyrosine release, systemic troponin-I), atrophy signaling pathways, and muscle phenotypic features and function were analyzed. In gastrocnemius of unloaded mice treated with resveratrol, body and muscle weight and function were attenuated, whereas muscle proteolysis (tyrosine release), proteolytic and apoptotic markers, atrophy signaling pathways, and myofiber CSA significantly improved. Resveratrol treatment of mice exposed to a seven-day period of unloading prevented body and muscle weight and limb strength loss, while an improvement in muscle proteolysis, proteolytic markers, atrophy signaling pathways, apoptosis, and muscle fiber CSA was observed in the gastrocnemius muscle. These findings may have potential therapeutic implications in the management of disuse muscle atrophy in clinical settings.

Voluntary Forelimbs Exercise Reduces Immobilization-Induced Mechanical Hyperalgesia in the Rat Hind Paw.

Voluntary exercise is sufficient to protect against neuropathic pain. However, it is unclear whether voluntary exercise reduces immobilization-induced hyperalgesia. We examined the effect of voluntary forelimb exercise on immobilized-induced hyperalgesia in hind paws of rats. Wistar rats were randomly divided into the (1) both hind limbs immobilized group (IM group), (2) immobilization and exercise with nonimmobilized fore limbs group (EX group), and (3) control group. In the IM and EX groups, the bilateral ankle joints of each rat were immobilized in full plantar flexion with a plaster cast for eight weeks. In the EX group, voluntary exercise using nonimmobilized forelimbs in the running wheel was administered during the immobilization period, while hind limbs were kept immobilized (60 min/day, 5 days/week). Mechanical hyperalgesia in the hind paw was measured using a digital von Frey device every week. To investigate the abnormality of primary sensory neurons and central sensitization, the number of calcitonin gene-related peptide-positive cells in the dorsal root ganglion and the expression level of calcitonin gene-related peptide in the spinal dorsal horn were analyzed by immunohistochemical staining. Immobilization-induced mechanical hyperalgesia was inhibited in the EX group compared to the IM group at three weeks after immobilization. In the EX group, the number of calcitonin gene-related peptide-positive cells in the dorsal root ganglion and the expression level of calcitonin gene-related peptide were significantly decreased compared to those in the IM group. Our results therefore suggest that voluntary forelimb exercise during hind limb immobilization partially reduces immobilization-induced hyperalgesia by suppressing that the plastic changes of the primary sensory nerves that excessively transmit pain and increased responsiveness of nociceptive neurons in the spinal dorsal horn.

Mitophagy reporter mouse analysis reveals increased mitophagy activity in disuse-induced muscle atrophy.

Muscle disuse induces atrophy through increased reactive oxygen species (ROS) released from damaged mitochondria. Mitophagy, the autophagic degradation of mitochondria, is associated with increased ROS production. However, the mitophagy activity status during disuse-induced muscle atrophy has been a subject of debate. Here, we developed a new mitophagy reporter mouse line to examine how disuse affected mitophagy activity in skeletal muscles. Mice expressing tandem mCherry-EGFP proteins on mitochondria were then used to monitor the dynamics of mitophagy activity. The reporter mice demonstrated enhanced mitophagy activity and increased ROS production in atrophic soleus muscles following a 14-day hindlimb immobilization. Results also showed an increased expression of multiple mitophagy genes, including Bnip3, Bnip3l, and Park2. Our findings thus conclude that disuse enhances mitophagy activity and ROS production in atrophic skeletal muscles and suggests that mitophagy is a potential therapeutic target for disuse-induced muscle atrophy.