Hindlimb Immobilization Research Articles

Cast immobilization of hindlimb upregulates sarcolipin expression in atrophied skeletal muscles and increases thermogenesis in C57BL/6J mice.

Mechanical unloading impairs cytosolic calcium (Ca2+) homeostasis in skeletal muscles. In this study, we investigated whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) itself or one of the regulators of the Ca2+ SERCA pump, sarcolipin (SLN), is altered to deregulate Ca2+ homeostasis in cast immobilized, atrophied muscles. Hindlimb muscles of 8-wk-old male C57BL/6J mice were subjected to bilateral cast immobilization for 2 wk. Two-week-cast immobilization induced both body weight and skeletal muscle loss. Highly phosphorylated Ca2+/calmodulin-dependent protein kinase II in the atrophied muscles suggested that cytosolic Ca2+ concentration was elevated. Extremely high expression levels of SLN mRNA and protein were observed in the atrophied muscles. Upregulation of SLN at the transcriptional level was supported by low RCAN1 expression, which is a negative regulator of SLN. We treated C2C12 cells with dexamethasone to mimic muscle atrophy in vitro and showed a direct relationship between high SLN mRNA expression and low Ca2+ uptake by sarcoplasmic reticulum. Since SLN reportedly plays a role in nonshivering thermogenesis, we performed a cold tolerance test of the whole body. As a result, we found that mice with cast immobilization showed high cold tolerance, suggesting that cast immobilization promoted whole body thermogenesis. Although the activity level was decreased during cast immobilization without change in food intake, adipose tissue weights also decreased significantly after cast immobilization. Concomitantly, we conclude that cast immobilization of hindlimb increased thermogenesis in C57Bl/6J mice, probably via high expression of SLN.

Effects of treadmill running and limb immobilization on knee cartilage degeneration and locomotor joint kinematics in rats following knee meniscal transection

This study examined the effects of reduced and elevated weight bearing on post-traumatic osteoarthritis (PTOA) development, locomotor joint kinematics, and degree of voluntary activity in rats following medial meniscal transection (MMT). Twenty-one adult rats were subjected to MMT surgery of the left hindlimb and then assigned to one of three groups: (1) regular (i.e., no intervention), (2) hindlimb immobilization, or (3) treadmill running. Sham surgery was performed in four additional rats. Voluntary wheel run time/distance was measured, and 3D hindlimb kinematics were quantified during treadmill locomotion using biplanar radiography. Rats were euthanized 8weeks after MMT or sham surgery, and the microstructure of the tibial cartilage and subchondral bone was quantified using contrast enhanced micro-CT. All three MMT groups showed signs of PTOA (full-thickness lesions and/or increased cartilage volume) compared to the sham group, however the regular and treadmill-running groups had greater osteophyte formation than the immobilization group. For the immobilization group, increased volume was only observed in the anterior region of the cartilage. The treadmill-running group demonstrated a greater knee varus angle at mid-stance than the sham group, while the immobilization group demonstrated greater reduction in voluntary running than all the other groups at 2 weeks post-surgery. Elevated weight-bearing via treadmill running at a slow/moderate speed did not accelerate PTOA in MMT rats when compared to regular weight-bearing. Reduced weight-bearing via immobilization may attenuate overall PTOA but still resulted in regional cartilage degeneration. Overall, there were minimal differences in hindlimb kinematics and voluntary running between MMT and sham rats.

Dietary Alaska pollack protein improves skeletal muscle weight recovery after immobilization-induced atrophy in rats.

The promotion of muscle recovery after immobilization is important to preserve an optimum health status. Here, we examined the effect of dietary Alaska pollack protein (APP) on skeletal muscle weight after atrophy induced by hind limb immobilization using plaster immobilization technique. Rat left limb was casted with a wetted plaster cast under anesthesia. After 2 weeks of feeding, the cast was removed and the rats were divided into three groups, namely, a baseline group, high-fat casein diet group, and high-fat APP diet group. After 3 weeks of feeding, the skeletal muscles (soleus, extensor digitorum longus [EDL], and gastrocnemius) were sampled. The estimated weight gains of soleus, gastrocnemius, and EDL muscle in the immobilized limbs were significantly larger in the rats fed with APP diet as compared with those fed with casein diet. In soleus muscle, dietary APP increased the expression of Igf1 and Myog genes in the immobilized limbs after the recovery period.

Morphological changes of the nerve after restorative treatment using electric welding technology (experimental study)

Objective – to study morphological features regenerative nerve neuroma after a complete intersection and restorative surgery with the use of electric welding technology.Materials and methods. The experimental model of operative intervention on injured peripheral nerve by immediate nerve stump’s epineurium welding and partial hindlimb immobilization was developed. The study was performed on 66 white, non-breeding male rats aged 5–7 months with a body weight of 350–450 g of vivarium breeding in Institute of Neurosurgery named after acad. A.P. Romodanov NAMS of Ukraine. Animals were divided into four experimental groups: 1 – surgical animals that included access to the sciatic nerve and its full intersection (n = 21), 2 – animals that were surgically intervened, which included access to the sciatic nerve, its intersection and the restoration of anatomical integrity by the application of an epineural suture (n = 18), 3 – animals that were operated surgically, which included access to the sciatic nerve, its intersection and the restoration of anatomical integrity by electric welding of the epineurium of the ends (n = 21), 4 – sham group, who were given access to the transverse nerve without its intersection (n = 6). In the experimental group of electric welding, the operation was performed using the apparatus EKWZ-300, «Patonmed» in the automatic mode dA2, with variable high-frequency (440 kHz) modulated current up to 0,3 A, the voltage at the 34 V pincer shield, the duration of the rectangular exposure pulse 0.8 s. Formated 5–6 electric weld point connections along the perimeter of the intersection with a complete alignment of the ends. The neurohistological method of study was used to study the morphological picture of the peripheral nerve in the region of the regenerative neurology and the distal segment in 1 and 3 months after surgical intervention. Results. It was established that simultaneous use of the technique of welded epineural joint of parts and partial immobilization of the limb in the postoperative period leads to uniform maturation of the neuro-connective tissue and its more complete neuroticisation.Conclusions. Recovery processes in the injured peripheral nerve occur better with the simultaneous formation of an immediate welded epineural joint of the ends and partial immobilization of the limb.

EIF3f depletion impedes mouse embryonic development, reduces adult skeletal muscle mass and amplifies muscle loss during disuse.

In muscular cells, eukaryotic initiation factor subunit f (eIF3f) activates protein synthesis by allowing physical interaction between mechanistic target of rapamycin complex 1 (MTORC1) and ribosomal protein S6 kinase 1 (S6K1), although its physiological role in animals is unknown. A knockout approach suggests that homozygous mice carrying a null mutation of the eIF3f gene fail to develop and consequently die at early embryonic stage, whereas heterozygous mice associated with a partial depletion of eIF3f gene grow normally and are phenotypically indistinguishable from wild-type mice. Heterozygous mice express reduced eIF3f mRNA and protein levels in skeletal muscles and show diminished muscle mass associated with a decrease in the protein synthesis rate and an inhibition of the MTORC1 pathway. During hindlimb immobilization, heterozygous eIF3f mice display an exacerbated immobilization-induced muscle atrophy associated with reduced protein synthesis. These results highlight the essential role of eIF3f during embryonic development and its involvement in muscular homeostasis via protein synthesis regulation. Eukaryotic translation initiation factor 3, subunit F (eIF3f), a component of eIF3 complex, plays an important role in protein synthesis regulation, although its physiological functions are unknown. We generated and analysed mice carrying a null mutation in the eIF3f gene. We showed that homozygous eIF3f knockout fail to develop and that eIF3f-/- embryos die at an early stage of development but after the pre-implantation stage. However, disrupting one eIF3f allele does not affect growth, viability and fertility of heterozygous mice but, instead, reduces eIF3f mRNA and protein levels in all tissues examined. Although heterozygous mice are phenotypically indistinguishable from wild-type mice, they present a diminished body weight and a lean mass reduction associated with normal body size. Interestingly, skeletal muscles are mainly affected and display an altered cell size without modification of fibre number. Skeletal muscles of heterozygous mice show a deficiency in polysome content, a decrease in protein synthesis rate and an inhibition of the mechanistic target of rapamycin (MTOR) pathway. We then studied the effects of hindlimb immobilization that mimic muscle disuse on heterozygous mice aiming to further explore the involvement of eIF3f in protein synthesis. We found that eIF3f partial depletion amplifies muscle atrophy compared to wild-type mice. Mass and cross-sectional area decreases were associated with reduced MTOR pathway activation and protein synthesis rate. Taken together, our data indicate that eIF3f is essential for mice embryonic development and controls adult skeletal muscle mass via protein synthesis regulation in a MTOR-dependent manner.

Pericyte transplantation improves skeletal muscle recovery following hindlimb immobilization.

Conditions of extended bed rest and limb immobilization can initiate rapid and significant loss of skeletal muscle mass and function. Physical rehabilitation is standard practice following a period of disuse, yet mobility may be severely compromised, and recovery is commonly delayed or incomplete in special populations. Thus, a novel approach toward recovery of muscle mass is highly desired. Pericytes [neuron-glial antigen 2 (NG2)+CD31-CD45- (Lineage- [Lin-]) and CD146+Lin-] demonstrate capacity to facilitate muscle repair, yet the ability to enhance myofiber growth following disuse is unknown. In the current study, 3-4-mo-old mice were unilaterally immobilized for 14 d (IM) or immobilized for 14 d followed by 14 d of remobilization (RE). Flow cytometry and targeted gene expression analyses were completed to assess pericyte quantity and function following IM and RE. In addition, a transplantation study was conducted to assess the impact of pericytes on recovery. Results from targeted analyses suggest minimal impact of disuse on pericyte gene expression, yet NG2+Lin- pericyte quantity is reduced following IM (P < 0.05). Remarkably, pericyte transplantation recovered losses in myofiber cross-sectional area and the capillary-to-fiber ratio following RE, whereas deficits remained with vehicle alone (P = 0.01). These findings provide the first evidence that pericytes effectively rehabilitate skeletal muscle mass following disuse atrophy.-Munroe, M., Dvoretskiy, S., Lopez, A., Leong, J., Dyle, M. C., Kong, H., Adams, C. M., Boppart, M. D. Pericyte transplantation improves skeletal muscle recovery following hindlimb immobilization.

Low-intensity muscle contraction exercise following the onset of arthritis improves hyperalgesia via reduction of joint inflammation and central sensitization in the spinal cord in a rat model

We examined the effect of immobilization, low-intensity muscle contraction exercise, and transcutaneous electrical nerve stimulation (TENS) on tissue inflammation and acute pain following the onset of arthritis in a rat model. Sixty Wistar rats were divided into five groups: (1) Arthritis group, (2) arthritis and immobilization (Immobilization group), (3) arthritis and low intensity muscle contraction (Exercise group), (4) arthritis and TENS (TENS group), and (5) sham arthritis (Sham group). Arthritis was induced in the right knee joints by single injection of 3% kaolin and carrageenan. Immobilization of the right hindlimb was conducted by full extension of the right knee joints and full plantar flexion of the ankle joints using a plaster cast for 7 days after injection. The right quadriceps muscles were subjected to electrical stimulation (frequency: 50 Hz; intensity: 2–3 mA) for 20 min/day as contraction exercise for one week. TENS was delivered at 20 min/day for one week (frequency: 50 Hz; intensity: 1 mA). The pressure pain threshold (PPT) and paw withdrawal response (PWR) were evaluated at 1 and 7 days after injection. We also analyzed the number of CD68-positive cells in the synovium by immunohistochemistry and determined the expression level of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn with immunofluorescence. Improvements of both PPT and PWR were observed in the Exercise group at 7 days after injection compared to those of the Arthritis and Immobilization groups, although only improvement of PPT was observed in the TENS group. The number of CD68-positive cells in the synovium and CGRP expression in the dorsal horn decreased only in the Exercise group. These results suggested that low-intensity muscle contraction exercise might be a better treatment for reduction of arthritis-induced inflammation and acute pain compared to immobilization and TENS.

The Effects of Dokhwalgisaeng-tang against Disuse Muscle Atrophy in Gastrocnemius of Rats

Background: The purpose of this study was to examine the effect of Dokhwalgisaeng-tang on immobilization-induced muscle atrophy. Methods: Twenty young male Sprague-Dawley rats were divided into 2 groups. The rats in Dokhwalgisaeng-tang group were orally administered Dokhwalgisaeng-tang water extract, and the rats in the control group were given saline only. Hind limb immobilization was performed with casting tape to keep the left ankle joint in a fully extended position. No intervention was performed on the right leg which was used as an intact region. After 2 weeks of immobilization, all animals were sacrificed, and the gastrocnemius muscle was dissected from both legs and weighed. The morphology of the right and the left gastrocnemius muscle in both the Dokhwalgisaeng-tang and the control group was assessed by hematoxylin and eosin staining. The muscle cross sectional area was examined by image analysis (Axiovision LE software). In addition, immunohistochemical staining was carried out using the free-floating method, and the number of apoptotic related proteins were counted (anti-BAX, anti-Bcl-2). Results: Dokhwalgisaeng-tang showed a significant protective effect against the reduction of the left gastrocnemius muscle (weight and muscle cross sectional area) compared with the control group. Moreover, the treatment with Dokhwalgisaeng-tang significantly reduced protein expression of BAX and increased protein expression of Bcl-2 in the gastrocnemius muscle compared with the control group. Conclusion: Dokhwalgisaeng-tang showed protective effects against disuse muscle atrophy, potentially through altered BAX and Bcl-2 protein expression in the gastrocnemius muscle.

Early movement restriction leads to maladaptive plasticity in the sensorimotor cortex and to movement disorders

Motor control and body representations in the central nervous system are built, i.e., patterned, during development by sensorimotor experience and somatosensory feedback/reafference. Yet, early emergence of locomotor disorders remains a matter of debate, especially in the absence of brain damage. For instance, children with developmental coordination disorders (DCD) display deficits in planning, executing and controlling movements, concomitant with deficits in executive functions. Thus, are early sensorimotor atypicalities at the origin of long-lasting abnormal development of brain anatomy and functions? We hypothesize that degraded locomotor outcomes in adulthood originate as a consequence of early atypical sensorimotor experiences that induce developmental disorganization of sensorimotor circuitry. We showed recently that postnatal sensorimotor restriction (SMR), through hind limb immobilization from birth to one month, led to enduring digitigrade locomotion with ankle-knee overextension, degraded musculoskeletal tissues (e.g., gastrocnemius atrophy), and clear signs of spinal hyperreflexia in adult rats, suggestive of spasticity; each individual disorder likely interplaying in self-perpetuating cycles. In the present study, we investigated the impact of postnatal SMR on the anatomical and functional organization of hind limb representations in the sensorimotor cortex and processes representative of maladaptive neuroplasticity. We found that 28 days of daily SMR degraded the topographical organization of somatosensory hind limb maps, reduced both somatosensory and motor map areas devoted to the hind limb representation and altered neuronal response properties in the sensorimotor cortex several weeks after the cessation of SMR. We found no neuroanatomical histopathology in hind limb sensorimotor cortex, yet increased glutamatergic neurotransmission that matched clear signs of spasticity and hyperexcitability in the adult lumbar spinal network. Thus, even in the absence of a brain insult, movement disorders and brain dysfunction can emerge as a consequence of reduced and atypical patterns of motor outputs and somatosensory feedback that induce maladaptive neuroplasticity. Our results may contribute to understanding the inception and mechanisms underlying neurodevelopmental disorders, such as DCD.

Local cyclical compression modulates macrophage function in situ and alleviates immobilization-induced muscle atrophy.

Physical inactivity gives rise to numerous diseases and organismal dysfunctions, particularly those related to aging. Musculoskeletal disorders including muscle atrophy, which can result from a sedentary lifestyle, aggravate locomotive malfunction and evoke a vicious circle leading to severe functional disruptions of vital organs such as the brain and cardiovascular system. Although the significance of physical activity is evident, molecular mechanisms behind its beneficial effects are poorly understood. Here, we show that massage-like mechanical interventions modulate immobilization-induced pro-inflammatory responses of macrophages in situ and alleviate muscle atrophy. Local cyclical compression (LCC) on mouse calves, which generates intramuscular pressure waves with amplitude of 50 mmHg, partially restores the myofiber thickness and contracting forces of calf muscles that are decreased by hindlimb immobilization. LCC tempers the increase in the number of cells expressing pro-inflammatory proteins, tumor necrosis factor-α and monocyte chemoattractant protein-1 (MCP-1), including macrophages in situ The reversing effect of LCC on immobilization-induced thinning of myofibers is almost completely nullified when macrophages recruited from circulating blood are depleted by administration of clodronate liposomes. Furthermore, application of pulsatile fluid shear stress, but not hydrostatic pressure, reduces the expression of MCP-1 in macrophages in vitro Together with the LCC-induced movement of intramuscular interstitial fluid detected by µCT analysis, these results suggest that mechanical modulation of macrophage function is involved in physical inactivity-induced muscle atrophy and inflammation. Our findings uncover the implication of mechanosensory function of macrophages in disuse muscle atrophy, thereby opening a new path to develop a novel therapeutic strategy utilizing mechanical interventions.

Regulation of muscle atrophy-related genes by the opposing transcriptional activities of ZEB1/CtBP and FOXO3.

Multiple physiopathological and clinical conditions trigger skeletal muscle atrophy through the induction of a group of proteins (atrogenes) that includes components of the ubiquitin–proteasome and autophagy-lysosomal systems. Atrogenes are induced by FOXO transcription factors, but their regulation is still not fully understood. Here, we showed that the transcription factor ZEB1, best known for promoting tumor progression, inhibits muscle atrophy and atrogene expression by antagonizing FOXO3-mediated induction of atrogenes. Compared to wild-type counterparts, hindlimb immobilization in Zeb1-deficient mice resulted in enhanced muscle atrophy and higher expression of a number of atrogenes, including Atrogin-1/Fbxo32, MuRF1/Trim63, Ctsl, 4ebp1, Gabarapl1, Psma1 and Nrf2. Likewise, in the C2C12 myogenic cell model, ZEB1 knockdown augmented both myotube diameter reduction and atrogene upregulation in response to nutrient deprivation. Mechanistically, ZEB1 directly represses in vitro and in vivo Fbxo32 and Trim63 promoter transcription in a stage-dependent manner and in a reverse pattern with MYOD1. ZEB1 bound to the Fbxo32 promoter in undifferentiated myoblasts and atrophic myotubes, but not in non-atrophic myotubes, where it is displaced by MYOD1. ZEB1 repressed both promoters through CtBP-mediated inhibition of FOXO3 transcriptional activity. These results set ZEB1 as a new target in therapeutic approaches to clinical conditions causing muscle mass loss.

Repressors of mTORC1 act to blunt the anabolic responseto feeding in the soleus muscle of a cast-immobilized mouse hindlimb.

We recently reported results showing that cast immobilization of a rat hindlimb rapidly leads to development of anabolic resistance as demonstrated by failure of oral leucine administration to activate the mechanistic target of rapamycin complex 1 (mTORC1) and stimulate protein synthesis in the soleus muscle. The goal of this study was to assess the possible contribution of several mTORC1 regulatory proteins to the development of anabolic resistance. To accomplish this, 14‐week‐old male C57BL/6 mice (n = 21) were subjected to unilateral cast immobilization of the hindlimb for either 1 or 3 days, and the immobilized limb was compared to its contralateral control. The mass of the soleus muscle was decreased in the immobilized compared to the non‐immobilized limb within 72‐h in association with diminished protein synthesis. In agreement with our previous report, a 24‐h casting period was sufficient to induce anabolic resistance, as demonstrated by blunted re‐feeding‐induced activation of mTORC1. Moreover, resistance of mTORC1 activation was associated not only with upregulated expression of REDD1, but also with altered expression of other mTORC1 regulatory proteins, that is, Sestrin1 and DEP domain‐containing mTOR interacting protein (DEPTOR). In addition, re‐feeding‐induced phosphorylation of DEPTOR was significantly impaired in the immobilized compared to the non‐immobilized limb. This work builds upon previous discoveries by our laboratory to elucidate the blunted mTORC1 response to stimuli during disuse of skeletal muscle induced by cast immobilization while highlighting new potential therapeutic targets for future countermeasures against muscle atrophy.

Quiet breathing in hindlimb casted mice

The hindlimb casting model was developed to study skeletal muscle reloading following a period of unloading. It is unknown if ventilation parameters of mice are affected by the casting model. We tested the hypothesis that hindlimb casted mice have similar ventilatory patterns compared to mice with the casts removed. Male CD-1 mice underwent 14 days of hindlimb immobilization via plaster casting. Breathing parameters were obtained utilizing unrestrained barometric plethysmography (UBP). Breathing traces were analyzed with Ponemah software for breathing frequency, tidal volume (TV), and minute ventilation (MV). Frequency, TV and MV did not show any differences in quiet breathing patterns during or post-casting in mice. Thus, the hindlimb casting model does not complicate breathing during and after casting and should not interfere with the unloading and reloading of skeletal muscle.

Immobilization rapidly induces thioredoxin-interacting protein gene expression together with insulin resistance in rat skeletal muscle.

Acute short duration of disuse induces the development of insulin resistance for glucose uptake in rodent skeletal muscle. Because thioredoxin-interacting protein (TXNIP) has been implicated in the downregulation of insulin signaling and glucose uptake, we examined the possibility that muscle disuse rapidly induces insulin resistance via increased TXNIP mRNA and protein expression. Male Wistar rats were subjected to unilateral 6-h hindlimb immobilization by plaster cast. At the end of this period, the soleus muscles from both immobilized and contralateral nonimmobilized hindlimbs were excised and examined. The 6-h immobilization resulted in an increase in TXNIP mRNA and protein expressions together with a decrease in insulin-stimulated 2-deoxyglucose uptake in the rat soleus muscle. Additionally, in the rats euthanized 6 h after the plaster cast removal, TXNIP protein expression and insulin-stimulated glucose uptake in the immobilized muscle had both been restored to a normal level. Various interventions (pretreatment with transcription inhibitor actinomycin D or AMP-dependent protein kinase activator 5-aminoimidazole-4-carboxamide ribonucleotide) also suppressed the increase in TXNIP protein expression in 6-h-immobilized muscle together with partial prevention of insulin resistance for glucose uptake. These results suggested the possibility that increased TXNIP protein expression in immobilized rat soleus muscles was associated with the rapid induction of insulin resistance for glucose uptake in that tissue. NEW & NOTEWORTHY The cellular mechanism by which disuse rapidly induces muscle insulin resistance for glucose uptake remains to be identified. Using a rat hindlimb immobilization model, our findings suggest the possibility that transcriptional upregulation of thioredoxin-interacting protein is associated with the immobilization-induced rapid development of insulin resistance in skeletal muscle.

Muscle regeneration potential and satellite cell activation profile during recovery following hindlimb immobilization in mice.

Reduced muscle activity leads to muscle atrophy and function loss in patients and animal models. Satellite cells (SCs) are postnatal muscle stem cells that play a pivotal role in skeletal muscle regeneration following injury. The regenerative potential, satellite cell numbers, and markers during recovery following immobilization of the hindlimb for 7 days were explored. In mice exposed to 7 days of hindlimb immobilization, in those exposed to recovery (7 days, splint removal), and in contralateral control muscles, muscle precursor cells were isolated from all hindlimb muscles (fluorescence-activated cell sorting, FACS) and SCs, and muscle regeneration were identified using immunofluorescence (gastrocnemius and soleus) and electron microscopy (EM, gastrocnemius). Expression of ki67, pax7, myoD, and myogenin was quantified (RT-PCR) from SC FACS yields. Body and grip strength were determined. Following 7 day hindlimb immobilization, a decline in SCs (FACS, immunofluorescence) was observed together with an upregulation of SC activation markers and signs of muscle regeneration including fusion to existing myofibers (EM). Recovery following hindlimb immobilization was characterized by a program of muscle regeneration events. Hindlimb immobilization induced a decline in SCs together with an upregulation of markers of SC activation, suggesting that fusion to existing myofibers takes place during unloading. Muscle recovery induced a significant rise in muscle precursor cells and regeneration events along with reduced SC activation expression markers and a concomitant rise in terminal muscle differentiation expression. These are novel findings of potential applicability for the treatment of disuse muscle atrophy, which is commonly associated with severe chronic and acute conditions.

Concurrent treatment with ursolic acid and low-intensity treadmill exercise improves muscle atrophy and related outcomes in rats.

The objective of this study was to analyze the concurrent treatment effects of ursolic acid (UA) and low-intensity treadmill exercise and to confirm the effectiveness of UA as an exercise mimetic to safely improve muscle atrophy-related diseases using Sprague-Dawley (SD) rats with skeletal muscle atrophy. Significant muscle atrophy was induced in male SD rats through hind limb immobilization using casting for 10 days. The muscle atrophy-induced SD rats were group into four: SED, sedentary; UA, daily intraperitoneal UA injection, 5 mg/kg; EX, low-intensity (10–12 m/min, 0° grade) treadmill exercise; and UEX, daily intraperitoneal UA injection, 5 mg/kg, and low-intensity (10–12 m/min, 0° grade) treadmill exercise. After 8 weeks of treatment, endurance capacity was analyzed using a treadmill, and tissues were extracted for analysis of visceral fat mass, body weight, muscle mass, expression of muscle atrophy- and hypertrophy-related genes, and endurance capacity. Although the effects of body weight gain control, muscle mass increase, and endurance capacity improvement were inadequate in the UA group, significant results were confirmed in the UEX group. The UEX group had significantly reduced body weight and visceral fat, significantly improved mass of tibialis anterior and gastrocnemius muscles, and significantly decreased atrophy-related gene expression of MuRF1 and atrogin-1, but did not have significant change in hypertrophy-related gene expression of Akt and mTOR. The endurance capacity was significantly improved in the EX and UEX groups. These data suggest that concurrent treatment with low-intensity exercise and UA is effective for atrophy-related physical dysfunctions.

An herbal formula consisting of Schisandra chinensis (Turcz.) Baill, Lycium chinense Mill and Eucommia ulmoides Oliv alleviates disuse muscle atrophy in rats

Ethnopharmacological relevanceSchisandra chinensis (Turcz.) Baill (SC), Lycium chinense Mill (LC) and Eucommia ulmoides Oliv (EU) are representative tonic herbal medicines that help to strengthen body muscles and bones making them stronger according to the Donguibogam, a tradition medical book of the Joseon Dynasty in Korea. Aim of the studyTo evaluate effects of an herbal formula consisting of SC, LC and EU on muscle atrophy in C2C12 myotubes and in a rat model of immobilization-induced muscle atrophy. Materials and methodsMuscle atrophy was developed by cast immobilization of unilateral hindlimb on rats for 3 weeks. Treatments were administered orally 14 times over 3 weeks. After treatments, we compared the change of body weight, muscle weight, grip strength, muscle fiber size, muscle fiber type shift by Grip strength meter, H&E stain and ATPase stain. And western blot was used for evaluating molecular mechanism in muscle atrophy on C2C12 cells. ResultsWhen taken individually, SC was the most effective of the three in inhibiting tumor necrosis factor alpha (TNF-α)-induced degeneration of C2C12 myogenesis. The formulation with a mass ratio of 2:1:1 SC: LC: EU (SSLE) was more effective against TNF-α-induced muscle atrophy than was a 1:1:1 SC: LC: EU (SLE) formula or any of the single herbal extracts. In a rat model of disuse muscle atrophy, the SSLE formula significantly inhibited reductions in muscle weight, grip strength and muscle fiber size induced by hindlimb immobilization, in a dose-dependent manner. The formula also inhibited immobilization-induced shifting of the muscle fiber type in soleus muscle. Treatment with SSLE inhibited TNF-α-induced expression of the atrogenes atrogin-1 and muscle RING-finger protein 1 in C2C12 cells. The SSLE formula also increased myoblast differentiation markers (myoD and myogenin) and activation of the Akt and mammalian target of rapamycin (mTOR) signaling pathway. ConclusionThese findings suggest that the SSLE formula prevents muscle atrophy through inhibition of the ubiquitin-proteasome system as well as upregulation of myoblast differentiation and muscle protein synthesis in C2C12 cells. Taken together, we conclude that the SSLE formula is invaluable for the development of therapeutic medicines to prevent disuse muscle atrophy and its accompanying muscle weakness.

Preventive Effects of the Dietary Intake of Medium-chain Triacylglycerols on Immobilization-induced Muscle Atrophy in Rats.

Previous studies have shown that medium-chain triacylglycerols (MCTs) exert favorable effects on protein metabolism. This study evaluated the effects of the dietary intake of MCTs on rat skeletal muscle mass and total protein content during casting-induced hindlimb immobilization, which causes substantial protein degradation and muscle atrophy. Rats were fed a standard diet containing long-chain triacylglycerols (LCTs) or MCTs for 3 days and then a unilateral hindlimb was immobilized while they received the same diet. After immobilization for 3, 7, and 14 days, muscle mass and total protein content in immobilized soleus muscle in the LCT-fed rats had markedly decreased compared to the contralateral muscle; however, these losses were partially suppressed in MCT-fed rats. Autophagosomal membrane proteins (LC-I and -II), which are biomarkers of autophagy-lysosome activity, did not differ significantly between the LCT- and MCT-fed rats. In contrast, the immobilization-induced increase in muscle-specific E3 ubiquitin ligase MuRF-1 protein expression in immobilized soleus muscle relative to contralateral muscle was completely blocked in the MCT-fed rats and was significantly lower than that observed in the LCT-fed rats. Collectively, these results indicate that the dietary intake of MCTs at least partly alleviates immobilization-induced muscle atrophy by inhibiting the ubiquitin-proteasome pathway.

Soluble Milk Proteins Improve Muscle Mass Recovery after Immobilization-Induced Muscle Atrophy in Old Rats but Do not Improve Muscle Functional Property Restoration.

Effect of 3 different dairy protein sources on the recovery of muscle function after limb immobilization in old rats. Longitudinal animal study. Institut National de la Recherche Agronomique (INRA). The study took part in a laboratory setting. Old rats were subjected to unilateral hindlimb immobilization for 8 days and then allowed to recover with 3 different dietary proteins: casein, soluble milk proteins or whey proteins for 49 days. Body weight, muscle mass, muscle fibre size, isometric, isokinetic torque, muscle fatigability and muscle oxidative status were measured before and at the end of the immobilization period and during the recovery period i.e 7, 21, 35 and 49 days post immobilization. In contrast to the casein diet, soluble milk proteins and whey proteins were efficient to favor muscle mass recovery after cast immobilization during aging. By contrast, none of the 3 diary proteins was able to improve muscle strength, power and fatigability showing a discrepancy between the recovery of muscle mass and function. However, the soluble milk proteins allowed a better oxidative capacity in skeletal muscle during the rehabilitation period. Whey proteins and soluble milk proteins improve muscle mass recovery after immobilization-induced muscle atrophy in old rats but do not allow muscle functional property restoration.

Effect of Bone Marrow Mesenchymal Stem Cells on Satellite Cell Proliferation and Apoptosis in Immobilization-Induced Muscle Atrophy in Rats.

BackgroundMuscle atrophy due to disuse occurs along with adverse physiological and functional changes, but bone marrow stromal cells (MSCs) may be able to act as muscle satellite cells to restore myofibers. Thus, we investigated whether MSCs could enhance the proliferation of satellite cells and suppress myonuclear apoptosis during immobilization.Material/MethodsWe isolated, purified, amplified, and identified MSCs. Rats (n=48) were randomized into 3 groups: WB group (n=16), IM-PBS group (n=16), and IM-MSC (n=16). Rat hind limbs were immobilized for 14 d, treated with MSCs or phosphate-buffered saline (PBS), and then studied using immunohistochemistry and Western blot analysis to characterize the proteins involved. Apoptosis was assessed by terminal deoxynucleotidyl transferase (TdT)-mediated deoxy-UTP nick end labeling (TUNEL) method.ResultsWe compared muscle mass, cross-sectional areas, and peak tetanic forces and noted insignificant differences between PBS- and MSC-treated animals, but satellite cell proliferation was significantly greater after MSC treatment (p<0.05). Apoptotic myonuclei were reduced (p<0.05) after MSC treatment as well. Pro-apoptotic Bax was down-regulated and anti-apoptotic Bcl-2 and p-Akt protein were upregulated (p<0.05).ConclusionsMSCs injected during hind limb immobilization can maintain satellite cell activity by suppressing myonuclear apoptosis.