CRISPR/Cas9-Mediated miR-29b Editing as a Treatment of Different Types of Muscle Atrophy in Mice.

Muscle atrophy is the loss of skeletal muscle mass and strength in response to diverse catabolic stimuli. At present, no effective treatments except exercise have been shown to reduce muscle atrophy clinically. Here, we report that CRISPR/Cas9-mediated genome editing through local injection into gastrocnemius muscles or tibialis anterior muscle efficiently targets the biogenesis processing sites in pre-miR-29b. In vivo, this CRISPR-based treatment prevented the muscle atrophy induced by angiotensin II (AngII), immobilization, and denervation via activation of the AKT-FOXO3A-mTOR signaling pathway and protected against AngII-induced myocyte apoptosis in mice, leading to significantly increased exercise capacity. Our work establishes CRISPR/Cas9-based gene targeting on miRNA as a potential durable therapy for the treatment of muscle atrophy and expands the strategies available interrogating miRNA function in vivo.

Hyperbaric Oxygen Therapy Attenuates Burn-Induced Denervated Muscle Atrophy.

Background: Neuronal apoptosis and inflammation in the ventral horn of the spinal cord contribute to denervated muscle atrophy post-burn. Hyperbaric oxygen therapy (HBOT) exerts anti-inflammation and neuroprotection. Furthermore, hypoxia-inducible factor (HIF)-1α has been reported to promote inflammation and apoptosis. We investigated the therapeutic potential of HBOT and the role of HIF-1α post-burn. Methods: Sprague-Dawley rats were divided into three groups: a control group, an untreated burn group receiving burn and sham treatment, and a HBOT group receiving burn injury and HBOT. The burn injury was induced with 75ºC ± 5ºC at the right hindpaw. HBOT (100% oxygen at 2.5 atmosphere, 90 min/day) and sham HBOT (21% oxygen at 1 atmosphere, 90 min/day) was started on day 28 after burn injury and continued for 14 treatments (days 28-41). Incapacitance (hind limb weight bearing) testing was conducted before burn and weekly after burn. At day 42 post-burn, the gastrocnemius muscle and the spinal cord ventral horn were analyzed. Results: HBOT improved burn-induced weight bearing imbalance. At day 42 post-burn, less gastrocnemius muscle atrophy and fibrosis were noted in the HBOT group than in the untreated burn group. In the ventral horn, HBOT attenuated the neuronal apoptosis and glial activation post-burn. The increases in phosphorylated AKT/mTOR post-burn were reduced after HBOT. HBOT also inhibited HIF-1α signaling, as determined by immunofluorescence and western blot. Conclusions: HBOT reduces burn-induced neuronal apoptosis in the ventral horn, possibly through HIF-1α signaling.

Efficacy of electrical stimulation of denervated muscle: A multicenter, double-blind, randomized clinical trial.

BACKGROUND: This was a multicenter, double-blind, randomized clinical trial to investigate the efficacy of electrical stimulation of denervated muscle (ESDM) on recovery of patients with peripheral nerve injuries. METHODS: We enrolled 38 patients with traumatic peripheral nerve injuries with axonal damage and clinical impairment of two muscles, who were randomly treated with real or sham electrical stimulation (ES). Clinical and neurophysiological examinations were performed before treatment, at the end of treatment, and 3 mo posttreatment, by the same physician who was blinded to the ES allocation. RESULTS: All patients improved but there was no significant beneficial effect of ESDM compared with sham treatment. CONCLUSIONS: This study failed to demonstrate the efficacy of ESDM for peripheral nerve injuries. However, given the large number of variables related to ES and the heterogeneity in disease etiologies and clinical manifestations, future studies on homogeneous populations using different stimulation protocols may be useful.

Natural History of Mixed and Motor Nerve Cryoablation in Humans-A Cohort Analysis.

Eight patients who underwent percutaneous cryoablation of mixed and/or motor nerves over a period of 5 years were identified. Distances from the ablation sites to origins of distal musculature were measured, and times to initial clinical recovery were collected. Strength progression over time following muscle activation was also collected and analyzed. All patients demonstrated activation of all muscles distal to the ablation, and the calculated mean rate of nerve regeneration based on distance to the origin of the assessed musculature and time to muscle activation for the group was 1.5 mm/day ± 1.1.

Low-Frequency Electrical Stimulation of Denervated Skeletal Muscle Retards Muscle and Trabecular Bone Loss in Aged Rats.

Electrical stimulation (ES)-induced muscle contraction has multiple effects; however, mechano-responsiveness of bone tissue declines with age. Here, we investigated whether daily low-frequency ES-induced muscle contraction treatment reduces muscle and bone loss and ameliorates bone fragility in early-stage disuse musculoskeletal atrophy in aged rats. Twenty-seven-month-old male rats were assigned to age-matched groups comprising the control (CON), sciatic nerve denervation (DN), or DN with direct low-frequency ES (DN+ES) groups. The structural and mechanical properties of the trabecular and cortical bone of the tibiae, and the morphological and functional properties of the tibialis anterior (TA) muscles were assessed one week after DN. ES-induced muscle contraction force mitigated denervation-induced muscle and trabecular bone loss and deterioration of the mechanical properties of the tibia mid-diaphysis, such as the stiffness, but not the maximal load, in aged rats. The TA muscle in the DN+ES group showed significant improvement in the myofiber cross-sectional area and muscle force relative to the DN group. These results suggest that low-frequency ES-induced muscle contraction treatment retards trabecular bone and muscle loss in aged rats in early-stage disuse musculoskeletal atrophy, and has beneficial effects on the functional properties of denervated skeletal muscle.

Dietary Genistein Prevents Denervation-Induced Muscle Atrophy in Male Rodents via Effects on Estrogen Receptor-α.

BACKGROUND: Genistein has high estrogenic activity. Previous studies have shown beneficial effects of estrogen or hormone replacement therapy on muscle mass and muscle atrophy. OBJECTIVE: We investigated the preventive effects and underlying mechanisms of genistein on muscle atrophy. METHODS: In Expt. 1, male Wistar rats were fed a diet containing no genistein [control (CON)] or 0.05% genistein (GEN; wt:wt diet) for 24 d. On day 14, the sciatic nerve in the left hind leg was severed, and the right hind leg was sham-treated. In Expt. 2, male C57BL6J mice were subcutaneously administered a vehicle (Veh group) or the estrogen receptor (ER) antagonist ICI 182,780 (ICI group) via an osmotic pump for 27 d, and each group was subsequently fed CON or GEN diets from day 3 to day 27. Muscle atrophy was induced on day 17 as in Expt. 1. In Expt. 3, male C57BL6J mice were subcutaneously administered vehicle or a selective ER agonist-ER-α [4,4',4'-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT)] or ER-ß [2,3-bis(4-hydroxyphenyl)-propionitrile (DPN)]-or genistein (GEN-sc-i) via an osmotic pump for 13 d, and muscle atrophy was induced on day 3 as in Expt. 1. The ratio of denervated soleus muscle weight to sham-operated soleus muscle weight (d/s ratio) was used as the index of muscle atrophy. RESULTS: Expt. 1: The d/s ratio in the GEN group was 20% higher than that in the CON group (P < 0.05). Expt. 2: The d/s ratio in the Veh-GEN group was 14% higher than that in the Veh-CON group (P < 0.05), although there was no significant difference between ICI-CON and ICI-GEN groups (P = 0.69). Expt. 3: The d/s ratio in the PPT-treated group was 20% greater than that in the Veh group (P < 0.05), but DPN and GEN-sc-i had no effect on the d/s ratio (P ≥ 0.05 compared with vehicle). CONCLUSION: Genistein intake mitigated denervation-induced soleus muscle atrophy. ER-α was related to the preventive effect of genistein on muscle atrophy.

Comparative decline of the protein profiles of nebulin in response to denervation in skeletal muscle.

The sliding filament model of the sarcomere was developed more than half a century ago. This model, consisting only of thin and thick filaments, has been efficacious in elucidating many, but not all, features of skeletal muscle. Work during the 1980s revealed the existence of two additional filaments: the giant filamentous proteins titin and nebulin. Nebulin, a giant myofibrillar protein, acts as a protein ruler to maintain the lattice arrays of thin filaments and plays a role in signal transduction and contractile regulation. However, the change of nebulin and its effect on thin filaments in denervation-induced atrophic muscle remains unclear. The purpose of this study is to examine the content and pattern of nebulin, myosin heavy chain (MHC), actin, and titin in innervated and denervated tibialis anterior (TA) muscles of rats using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), densitometry and electron microscopic (EM) analyses. The results revealed that denervation induced muscle atrophy is accompanied by decreased nebulin content in a time-dependent manner. For instant, the levels of nebulin in denervated muscles were markedly (P < 0.05) decreased, about 24.6% and 40.2% in comparison with innervated muscle after denervation of 28 and 56 days, respectively. The nebulin/MHC, nebulin/actin, and nebulin/titin ratios were decreased, suggesting a concomitant reduction of nebulin in denervated muscle. Moreover, a western blotting assay proved that nebulin declined faster than titin on 28 and 56 days of denervated muscle. In addition, EM study revealed that the disturbed arrangements of myofilaments and a disorganized contractile apparatus were also observed in denervated muscle. Overall, the present study provides evidence that nebulin is more sensitive to the effect of denervation than MHC, actin, and titin. Nebulin decline indeed resulted in disintegrate of thin filaments and shortening of sarcomeres.

Selective peripheral denervation for the treatment of spasmodic torticollis: long-term follow-up results from 648 patients.

BACKGROUND: Selective peripheral denervation (SPD) is currently the primary surgical treatment for spasmodic torticollis (ST). Our objective here is to report on the outcome of patients treated with this procedure for ST in our department. METHODS: Between June 1995 and June 2013, 648 patients underwent SPD for ST. We included 293 women (45.2 %) and 355 men (54.8 %) with a mean age of 41.1 years (range, 8-74 years) at the onset of dystonia. Surgery was performed at a mean of 3.6 years (range, 1-32 years) after onset of symptoms. Data on clinical presentation, radiological studies, operation tragedy, clinical outcomes and complications were analysed retrospectively. For evaluation of clinical outcomes, patients' responses were assessed using the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS). RESULTS: Results were obtained from all 648 patients with a follow-up period ranging from 11 months to 154 months (mean, 33.4 months). The mean preoperative TWSTRS score was 54.7 ± 18.3 points (range, 39-67 points), which decreased to 31.1 ± 11.6 points postoperatively (range, 1-67 points); a significant improvement was observed between preoperative and postoperative TWSTRS evaluation; the clinical improvement of TWSTRS was 73.5 ± 11.9 %. In addition, no deaths and serious complications occurred in this cohort of patients. CONCLUSIONS: SPD is an effective surgical method for patients with ST. This procedure should be recommended if conservative therapy does not offer satisfactory relief of symptoms.

Soluble Factors Associated with Denervation-induced Skeletal Muscle Atrophy.

Skeletal muscle tissue has the critical function of mechanical support protecting the body. In addition, its functions are strongly influenced by the balanced synthesis and degradation processes of structural and regulatory proteins. The inhibition of protein synthesis and/or the activation of catabolism generally determines a pathological state or condition called muscle atrophy, a reduction in muscle mass that results in partial or total loss of function. It has been established that many pathophysiological conditions can cause a decrease in muscle mass. Skeletal muscle innervation involves stable and functional neural interactions with muscles via neuromuscular junctions and is essential for maintaining normal muscle structure and function. Loss of motor innervation induces rapid skeletal muscle fiber degeneration with activation of atrophy-related signaling and subsequent disassembly of sarcomeres, altering normal muscle function. After denervation, an inflammation stage is characterized by the increased expression of pro-inflammatory cytokines that determine muscle atrophy. In this review, we highlighted the impact of some soluble factors on the development of muscle atrophy by denervation.

Expression of mitochondrial fission and fusion regulatory proteins in skeletal muscle during chronic use and disuse.

INTRODUCTION: The mitochondrial network within cells is mediated by fission and fusion processes. METHODS: We investigated the expression of the proteins responsible for these events during conditions of altered oxidative capacity. RESULTS: With chronic contractile activity, the mitochondrial reticulum increased in size, along with concomitant increases in the fusion proteins Opa1 and Mfn2 (by 36% and 53%; P < 0.05). When we induced muscle disuse through denervation for 7 days, fragmented mitochondria were observed, along with significant decreases in the expression of Mfn2 and Opa1 (by 84% and 70%). To assess the effects of aging on mitochondrial morphology, young (5 month) and aged (35 month) Fisher 344 Brown Norway rats were used. Aged animals also possessed smaller mitochondria and displayed increased levels of fission proteins. CONCLUSIONS: Chronic muscle use increases the ratio of fusion:fission proteins, leading to reticular mitochondria, whereas muscle disuse and aging result in a decrease in this ratio, culminating in fragmented organelles.

Clenbuterol suppresses proteasomal and lysosomal proteolysis and atrophy-related genes in denervated rat soleus muscles independently of Akt.

Although it is well known that administration of the selective ß(2)-adrenergic agonist clenbuterol (CB) protects muscle following denervation (DEN), the underlying molecular mechanism remains unclear. We report that in vivo treatment with CB (3 mg/kg sc) for 3 days induces antiproteolytic effects in normal and denervated rat soleus muscle via distinct mechanisms. In normal soleus muscle, CB treatment stimulates protein synthesis, inhibits Ca(2+)-dependent proteolysis, and increases the levels of calpastatin protein. On the other hand, the administration of CB to DEN rats ameliorates the loss of muscle mass, enhances the rate of protein synthesis, attenuates hyperactivation of proteasomal and lysosomal proteolysis, and suppresses the transcription of the lysosomal protease cathepsin L and of atrogin-1/MAFbx and MuRF1, two ubiquitin (Ub) ligases involved in muscle atrophy. These effects were not associated with alterations in either IGF-I content or Akt phosphorylation levels. In isolated muscles, CB (10(-6) M) treatment significantly attenuated DEN-induced overall proteolysis and upregulation in the mRNA levels of the Ub ligases. Similar responses were observed in denervated muscles exposed to 6-BNZ-cAMP (500 µM), a PKA activator. The in vitro addition of triciribine (10 µM), a selective Akt inhibitor, did not block the inhibitory effects of CB on proteolysis and Ub ligase mRNA levels. These data indicate that short-term treatment with CB mitigates DEN-induced atrophy of the soleus muscle through the stimulation of protein synthesis, downregulation of cathepsin L and Ub ligases, and consequent inhibition of lysosomal and proteasomal activities and that these effects are independent of Akt and possibly mediated by the cAMP/PKA signaling pathway.

Electrical stimulation impairs early functional recovery and accentuates skeletal muscle atrophy after sciatic nerve crush injury in rats.

Neuromuscular recovery after peripheral nerve lesion depends on the regeneration of severed axons that re-establish their functional connection with the denervated muscle. The aim of this study was to determine the effects of electrical stimulation (ES) on the neuromuscular recovery after nerve crush injury in rats. Electrical stimulation was carried out on the tibialis anterior (TA) muscle after sciatic nerve crush injury in a rat model. Six ES sessions were administered every other day starting from day 3 postinjury until the end of the experiment (day 14). The sciatic functional index was calculated. Muscle excitability, neural cell adhesion molecule (N-CAM) expression, and muscle fiber cross-sectional area (CSA) were accessed from TA muscle. Regenerated sciatic nerves were analyzed by light and confocal microscopy. Both treated (crush+ES) and untreated (crush) groups had their muscle weight and CSA decreased compared with the normal group (P < 0.05). Electrical stimulation accentuated muscle fiber atrophy more in the crush+ES than in the crush group (P < 0.05). N-CAM expression increased in both crush and crush+ES groups compared with the normal group (P < 0.05). Regenerated nerves revealed no difference between the crush and crush+ES groups. Nevertheless, functional recovery at day 14 post-injury was significantly lower in crush+ES group compared with the crush group. In addition, the crush+ES group had chronaxie values significantly higher on days 7 and 13 compared with the crush group, which indicates a decrease in muscle excitability in the crush+ES animals. The results of this study do not support a benefit of the tested protocol of ES during the period of motor nerve recovery following injury.

Denervation drives skeletal muscle atrophy and induces mitochondrial dysfunction, mitophagy and apoptosis via miR-142a-5p/MFN1 axis.

Rationale: Peripheral nerve injury is common in clinic, which leads to severe atrophy and dysfunction of the denervated muscles, but the underlying mechanism is not fully understood. Recent studies advanced the causative role of mitochondrial dysfunction in muscle atrophy, while the upstream triggers remained unclear. Methods: In the present study, Atrophy of gastrocnemius and tibialis anterior (TA) were evaluated in mice sciatic nerve transection model. Transmission electron microscopy (TEM) was then used to observe the microstructure of atrophic gastrocnemius and mitochondria. Subsequently, small RNA sequencing, luciferase reporter assay and Electrophoretic Mobility Shift (EMSA) were performed to explore the potential signaling pathway involved in skeletal muscle atrophy. The effects of the corresponding pathway on mitochondrial function, mitophagy, apoptosis and muscle atrophy were further determined in C2C12 cells and denervated gastrocnemius. Results: Gastrocnemius and TA atrophied rapidly after denervation. Obvious decrease of mitochondria number and activation of mitophagy was further observed in atrophic gastrocnemius. Further, miR-142a-5p/ mitofusin-1 (MFN1) axis was confirmed to be activated in denervated gastrocnemius, which disrupted the tubular mitochondrial network, and induced mitochondrial dysfunction, mitophagy and apoptosis. Furthermore, the atrophy of gastrocnemius induced by denervation was relieved through targeting miR-142a-5p/MFN1 axis. Conclusions: Collectively, our data revealed that miR-142a-5p was able to function as an important regulator of denervation-induced skeletal muscle atrophy by inducing mitochondrial dysfunction, mitophagy, and apoptosis via targeting MFN1. Our findings provide new insights into the mechanism of skeletal muscle atrophy following denervation and propose a viable target for therapeutic intervention in individuals suffering from muscle atrophy after peripheral nerve injury.

Architectural Changes in the Medial Gastrocnemius on Sonography after Nerve Ablation in Healthy Adults.

Architectural changes in healthy muscle after denervation have not yet been reported. This study aimed to investigate architectural changes in the medial head of the gastrocnemius muscle (GCM) after aesthetic tibial nerve ablation in healthy adults using ultrasonography (US). The effects of tibial nerve ablation were verified by visual observation and surface electromyography analysis. US images of medial GCMs were taken by one trained physician using B-mode and real-time US with a linear-array probe before nerve ablation, at 1 week after nerve ablation and at 3 months after nerve ablation in an anatomic standing position with the feet about shoulder-width apart in 19 healthy adults (17 females and 2 males). Muscle thickness was significantly reduced on the left side at 1 week and 3 months after the procedure and on the right side at 3 months after the procedure (p<0.050). Although fascicle length was not significantly changed, pennation angle was significantly reduced on both sides at 3 months after the procedure (p<0.050). Muscle thickness and pennation angle of the muscle fascicle were significantly reduced, although fascicle length was not significantly changed, after tibial nerve ablation in the medial GCM of healthy adults.

Targeted ablation of the cellular inhibitor of apoptosis 1 (cIAP1) attenuates denervation-induced skeletal muscle atrophy.

BACKGROUND: Skeletal muscle atrophy is a pathological condition that contributes to morbidity in a variety of conditions including denervation, cachexia, and aging. Muscle atrophy is characterized as decreased muscle fiber cross-sectional area and protein content due, in part, to the proteolytic activities of two muscle-specific E3 ubiquitin ligases: muscle RING-finger 1 (MuRF1) and muscle atrophy F-box (MAFbx or Atrogin-1). The nuclear factor-kappa B (NF-κB) pathway has emerged as a critical signaling network in skeletal muscle atrophy and has become a prime therapeutic target for the treatment of muscle diseases. Unfortunately, none of the NF-κB targeting drugs are currently being used to treat these diseases, likely because of our limited knowledge and specificity, for muscle biology and disease. The cellular inhibitor of apoptosis 1 (cIAP1) protein is a positive regulator of tumor necrosis factor alpha (TNFα)-mediated classical NF-κB signaling, and cIAP1 loss has been shown to enhance muscle regeneration during acute and chronic injury. METHODS: Sciatic nerve transection in wild-type, cIAP1-null and Smac mimetic compound (SMC)-treated mice was performed to investigate the role of cIAP1 in denervation-induced atrophy. Genetic in vitro models of C2C12 myoblasts and primary myoblasts were also used to examine the role of classical NF-κB activity in cIAP1-induced myotube atrophy. RESULTS: We found that cIAP1 expression was upregulated in denervated muscles compared to non-denervated controls 14 days after denervation. Genetic and pharmacological loss of cIAP1 attenuated denervation-induced muscle atrophy and overexpression of cIAP1 in myotubes was sufficient to induce atrophy. The induction of myotube atrophy by cIAP1 was attenuated when the classical NF-κB signaling pathway was inhibited. CONCLUSIONS: These results demonstrate the cIAP1 is an important mediator of NF-κB/MuRF1 signaling in skeletal muscle atrophy and is a promising therapeutic target for muscle wasting diseases.

Overexpression of osteoactivin protects skeletal muscle from severe degeneration caused by long-term denervation in mice.

We have previously shown that osteoactivin, a type I membrane glycoprotein expressed in myofibers, upregulated expression of matrix metalloprotease (MMP)-3 and MMP-9 in fibroblasts infiltrated denervated skeletal muscle in mice. To address whether osteoactivin-mediated increase in MMPs in skeletal muscle is useful for regeneration of denervated skeletal muscle, we subjected osteoactivin-transgenic mice to long-term denervation for 70 or 90 days. Long-term denervation caused severe degeneration of myofibers and fibrosis in skeletal muscle of wild-type mice. However, overexpression of osteoactivin protected skeletal muscle from such changes. Infiltration of fibroblast-like cells and collagen deposition were sustained at low levels after long-term denervation in skeletal muscle of osteoactivin-transgenic mice. This cytoprotective effect of osteoactivin was supported by the expression of regeneration/degeneration-associated genes in the gastrocnemius muscle during denervation. Denervation significantly upregulated the expression of anti-fibrotic genes, such as glypican-1 and decorin-1, in the gastrocnemius muscle of osteoactivin-transgenic mice, compared with wild-type mice. In contrast, overexpression of osteoactivin caused a significant reduction in denervation-induced expression of elongation factor 1A-1, an indicator for the persistence of degenerated cells. Our results suggest that an osteoactivin-mediated increase in MMPs in skeletal muscle might be useful for protecting injured muscle from fibrosis, leading to full regeneration after denervation.

Electromyographic findings after different selective neck dissections.

OBJECTIVES: The objective was to compare electrophysiologic investigations of the upper trapezius muscle (UT) after different selective neck dissections (SND) and analyze the differences between types of SND and the preservation and excision of the cervical nerves (the C2-4 rami of the cervical plexus). STUDY DESIGN: Retrospective study of 54 patients (average age, 65.1 +/- 9.6 yr, 45 males) with 70 SND. METHODS: Patients underwent needle electromyography (EMG) of the UT by 4 months after surgery. The findings were rated according to the 5 point EMG scale system from 1 (total denervation: positive sharp wave or fibrillation potential at rest and electrical silence at voluntary contraction) to 5 (normal pattern). RESULTS: The average EMG scale was 1.7 +/- 1.1, 58.6% for score 1 and only 5.7% for score 5. There was not a significant difference in the EMG scale between the types of SND, whereas the group in which the cervical nerves were excised was significantly lower than in that in which it was preserved. The average EMG scales in the former and latter were 1.5 +/- 0.8 and 2.0 +/- 1.3, 68.8%. CONCLUSIONS: The study data confirm that complete or incomplete denervation of the UT was caused by axonal injury of the spinal accessory nerve, even though it was spared, because of traction of the nerve during neck dissection. Second, the excision of the C2 to 4 rami of the cervical plexus caused worse damage of the UT. It is suggested that it is important to preserve the cervical nerves to avoid denervation of the UT.

Acupuncture plus low-frequency electrical stimulation (Acu-LFES) attenuates denervation-induced muscle atrophy.

Muscle wasting occurs in a variety of clinical situations, including denervation. There is no effective pharmacological treatment for muscle wasting. In this study, we used a tibial nerve denervation model to test acupuncture plus low-frequency electric stimulation (Acu-LFES) as a therapeutic strategy for muscle atrophy. Acupuncture needles were connected to an SDZ-II electronic acupuncture device delivering pulses at 20 Hz and 1 mA; the treatment was 15 min daily for 2 wk. Acu-LFES prevented soleus and plantaris muscle weight loss and increased muscle cross-sectional area in denervated mice. The abundances of Pax7, MyoD, myogenin, and embryonic myosin heavy chain were significantly increased by Acu-LFES in both normal and denervated muscle. The number of central nuclei was increased in Acu-LFES-treated muscle fibers. Phosphorylation of Akt was downregulated by denervation leading to a decline in muscle mass; however, Acu-LFES prevented the denervation-induced decline largely by upregulation of the IGF-1 signaling pathway. Acu-LFES reduced the abundance of muscle catabolic proteins forkhead O transcription factor and myostatin, contributing to the attenuated muscle atrophy. Acu-LFES stimulated the expression of macrophage markers (F4/80, IL-1b, and arginase-1) and inflammatory cytokines (IL-6, IFNγ, and TNFα) in normal and denervated muscle. Acu-LFES also stimulated production of the muscle-specific microRNAs miR-1 and miR-206. We conclude that Acu-LFES is effective in counteracting denervation-induced skeletal muscle atrophy and increasing muscle regeneration. Upregulation of IGF-1, downregulation of myostatin, and alteration of microRNAs contribute to the attenuation of muscle atrophy in denervated mice.

Effects of denervation on neuromuscular junctions in the thyroarytenoid muscle.

OBJECTIVES/HYPOTHESIS: To evaluate the effects of denervation on muscle fibers and neuromuscular junctions (NMJ) of the rat thyroarytenoid (TA) muscle with a histochemical method to monitor the status of degenerative NMJ. STUDY DESIGN: Quantitative assessment to monitor the status of degenerative muscle fibers and NMJ in the TA muscle. METHODS: Wistar rats were killed at 6, 12, 18, and 24 hours and at 2, 4, and 10 weeks after left recurrent laryngeal nerve (RLN) transection. Hematoxylin-eosin stain was used to evaluate the atrophic changes of the TA muscle. The pre- and postsynaptic structures of the NMJ were detected histochemically. These changes were evaluated by comparing the results between the treated (T) and untreated (U) sides (T/U ratio) in the same section. RESULTS: The atrophic changes in the TA muscle progressed gradually, and at 10 weeks, the T/U ratios of the entire muscle area and of the muscle fiber size decreased to 53.2 +/- 10.7% and 55.5 +/- 6.8%, respectively (P < .01). The number of nerve terminals decreased significantly at 18 hours (P < .01), and they disappeared completely by 24 hours. In contrast, at 10 weeks, 70.5 +/- 12.4% (P < .01) of acetylcholine receptors (AchRs) were preserved. CONCLUSIONS: In the rat TA muscle, denervation influences the presynaptic nerve terminals more than the postsynaptic AchRs and the muscle fibers. The results could be a basis for understanding the mechanism of laryngeal denervation and reinnervation processes in animal models.

Long-term denervation in humans causes degeneration of both contractile and excitation-contraction coupling apparatus, which is reversible by functional electrical stimulation (FES): a role for myofiber regeneration?

Over the last 30 years there has been considerable interest in the use of functional electrical stimulation (FES) to restore movement to the limbs of paralyzed patients. Spinal cord injury causes a rapid loss in both muscle mass and contractile force. The atrophy is especially severe when the injury involves lower motoneurons because many months after spinal cord injury, atrophy is complicated by fibrosis and fat substitution. In this study we describe the effects of long-term lower motoneuron denervation of human muscle and present the structural results of muscle trained using FES. By means of an antibody for embryonic myosin, we demonstrate that many regenerative events continue to spontaneously occur in human long-term denervated and degenerated muscle (DDM). In addition, using electron microscopy, we describe i) the overall structure of fibers and myofibrils in long-term denervated and degenerated muscle, including the effects of FES, and ii) the structure and localization of calcium release units, or triads; the structures reputed to activate muscle contraction during excitation-contraction coupling (ECC). Both apparatus undergo disarrangement and re-organization following long-term denervation and FES, respectively. The poor excitability of human long-term DDM fibers, which extends to the first periods of FES training, may be explained in terms of the spatial disorder of the ECC apparatus. Its disorganization and re-organization following long-term denervation and FES, respectively, may play a key role in the parallel disarrangement and re-organization of the myofibrils that characterize denervation and FES training. The present structural studies demonstrate that the protocol used during FES training is effective in reverting long-term denervation atrophy and dystrophy. The mean fiber diameter in FES biopsies is 42.2 +/- 14.8 SD (p < 0.0001 vs DDM 14.9 +/- 6.0 SD); the mean percentile of myofiber area of the biopsy is 94.3 +/- 5.7 SD (p < 0.0001 vs DDM 25.7 +/- 23.7 SD); the mean percentile fat area is 2.1 +/- 2.4 SD (p < 0.001 vs DDM 12.8 +/- 12.1 SD); and the mean percentile connective tissue area is 3.6 +/- 4.6 SD (p < 0.001 vs DDM 61.6 +/- 20.1 SD). In DDM biopsies more than 50% of myofibers have diameter smaller than 10 microm, while the FES-trained subjects have more that 50% of myofibers larger than 30 microm. The recovery of muscle mass seems to be the result of both a size increase of the surviving fibers and the regeneration of new myofibers.