Butyrate prevents muscle atrophy after sciatic nerve crush

Introduction: Histone deacetylases (HDACs) have been implicated in neurogenic muscle atrophy, but the mechanisms by which HDAC inhibitors might have beneficial effects are not defined. Methods: We used sciatic nerve crush to determine the effect of butyrate on denervation‐induced gene expression and oxidative stress. Results: Butyrate treatment initiated 3 weeks before injury and continued 1 week after injury increases histone acetylation and reduces muscle atrophy after nerve crush. Butyrate delivered only after nerve crush similarly prevented muscle atrophy. Butyrate had no effect on the increase in histone deacetylase 4 (HDAC4) protein levels following nerve crush but prevented the increase in expression of myogenin, MuRF1, and atrogin‐1. Butyrate did not affect mitochondrial reactive oxygen species production, but it increased antioxidant enzyme activity, reduced proteasome activity, and reduced oxidative damage following nerve injury. Conclusions: These data suggest that HDAC inhibitors are promising pharmacological agents for treating neurogenic muscle atrophy. Muscle Nerve 52: 859–868, 2015     

Role of ubiquitin–proteasome proteolysis in muscle fiber destruction in experimental chloroquine‐induced myopathy

Previous studies have documented the presence of rimmed vacuoles, atrophic fibers, and increased lysosomal cathepsin activity in skeletal muscle from animal models of chloroquine‐induced myopathy, suggesting that muscle fibers in this type of myopathy may be degraded via the lysosomal‐proteolysis pathway. Given recent evidence of abnormal ubiquitin accumulation in rimmed vacuoles, in this study we chose to examine the significance of the ubiquitin–proteasome proteolytic system in the process of muscle fiber destruction in experimental chloroquine myopathy. Expression of ubiquitin, 26S proteasome proteins, and ubiquitin ligases, such as muscle‐specific RING finger‐1 (MuRF‐1) and atrogin‐1/muscle atrophy F‐box protein (MAFbx), was analyzed in innervated and denervated rat soleus muscles after treatment with either saline or chloroquine. Abnormal accumulation of rimmed vacuoles was observed only in chloroquine‐treated denervated muscles. Ubiquitin and proteasome immunostaining, and ubiquitin, MuRF‐1, and atrogin‐1/MAFbx mRNAs were significantly increased in denervated soleus muscles from saline‐ and chloroquine‐treated rats when compared with contralateral innervated muscles. Further, ubiquitin and ubiquitin ligase mRNA levels were higher in denervated muscles from chloroquine‐treated rats when compared with saline‐treated rats. These data demonstrate increased proteasomes and ubiquitin in denervated muscles from chloroquine‐treated rats and suggest that the ubiquitin–proteasome proteolysis pathway as well as the lysosomal‐proteolysis pathway mediate muscle fiber destruction in experimental chloroquine myopathy. Muscle Nerve 39: 521–528, 2009     

Prevention of muscle disuse atrophy by MG132 proteasome inhibitor

Introduction: Our goal was to determine whether in vivo administration of the proteasome inhibitor MG132 can prevent muscle atrophy caused by hindlimb unloading (HU). Methods: Twenty‐seven NMRI mice were assigned to a weight‐bearing control, a 6‐day HU, or a HU+MG132 (1 mg/kg/48 h) treatment group. Results: Gastrocnemius wasting was significantly less in HU+MG132 mice (?6.7 ± 2.0%) compared with HU animals (?12.6 ± 1.1%, P = 0.011). HU was also associated with an increased expression of MuRF‐1 (P = 0.006), MAFbx (P = 0.001), and USP28 (P = 0.027) mRNA, whereas Nedd4, E3α, USP19, and UBP45 mRNA did not change significantly. Increases in MuRF‐1, MAFbx, and USP28 mRNA were largely repressed after MG132 administration. β5 proteasome activity tended to increase in HU (+16.7 ± 6.1%, P = 0.086). Neither β1 and β2 proteasome activities nor ubiquitin‐conjugated proteins were changed by HU. Conclusions: Our results indicate that in vivo administration of MG132 partially prevents muscle atrophy associated with disuse and highlight an unexpected regulation of MG132 proteasome inhibitor on ubiquitin‐ligases. Muscle Nerve, 2011     

Loss of sparc in mouse skeletal muscle causes myofiber atrophy

Introduction: The expression of secreted protein acidic and rich in cysteine (SPARC) in skeletal muscle decreases with age. Here, we examined the role of SPARC in skeletal muscle by reducing its expression. Methods: SPARC expression was suppressed by introducing short interfering RNA (siRNA) into mouse tibialis anterior muscle. Myofiber diameter, atrogin1, and muscle RING‐finger protein 1 (MuRF1) expression, and tumor necrosis factor‐α (TNFα) and transforming growth factor‐β (TGFβ) signaling were then analyzed. Results: Reduced SPARC expression caused decreases in the diameter of myofibers, especially fast‐type ones, accompanied by upregulation of atrogin1, but not MuRF1, at 10 days after siRNA transfection. The expression of TNFα and TGFβ and the phosphorylation status of p38 were not affected by SPARC knockdown, whereas Smad3 phosphorylation was increased at 2 days after siRNA transfection. Conclusions: The loss of SPARC not only upregulates atrogin1 expression but also enhances TGFβ signaling, which may in turn cause muscle atrophy. Muscle Nerve 48:791–799, 2013     

Leucine attenuates skeletal muscle wasting via inhibition of ubiquitin ligases

The aim of this study was to assess the effect of leucine supplementation on elements of the ubiquitin–proteasome system (UPS) in rat skeletal muscle during immobilization. This effect was evaluated by submitting the animals to a leucine supplementation protocol during hindlimb immobilization, after which different parameters were determined, including: muscle mass; cross‐sectional area (CSA); gene expression of E3 ligases/deubiquitinating enzymes; content of ubiquitinated proteins; and rate of protein synthesis. Our results show that leucine supplementation attenuates soleus muscle mass loss driven by immobilization. In addition, the marked decrease in the CSA in soleus muscle type I fibers, but not type II fibers, induced by immobilization was minimized by leucine feeding. Interestingly, leucine supplementation severely minimized the early transient increase in E3 ligase [muscle ring finger 1 (MuRF1) and muscle atrophy F‐box (MAFbx)/atrogin‐1] gene expression observed during immobilization. The reduced peak of E3 ligase gene expression was paralleled by a decreased content of ubiquitinated proteins during leucine feeding. The protein synthesis rate decreased by immobilization and was not affected by leucine supplementation. Our results strongly suggest that leucine supplementation attenuates muscle wasting induced by immobilization via minimizing gene expression of E3 ligases, which consequently could downregulate UPS‐driven protein degradation. It is notable that leucine supplementation does not restore decreased protein synthesis driven by immobilization. Muscle Nerve, 2010     

Abnormalities of NBR1, a novel autophagy-associated protein,in muscle fibers of sporadic inclusion-body myositis

Intra-muscle fiber accumulation of ubiquitinated protein aggregates containing several conformationally modified proteins, including amyloid-β and phosphorylated tau, is characteristic of the pathologic phenotype of sporadic inclusion-body myositis (s-IBM), the most common progressive degenerative myopathy of older persons. Abnormalities of protein-degradation, involving both the 26S proteasome and autophagic-lysosomal pathways, were previously demonstrated in s-IBM muscle. NBR1 is a ubiquitin-binding scaffold protein importantly participating in autophagic degradation of ubiquitinated proteins. Whereas abnormalities of p62, a ubiquitin-binding protein, were previously described in s-IBM, abnormalities of NBR1 have not been reported in s-IBM. We have now identified in s-IBM muscle biopsies that NBR1, by: (a) immunohistochemistry, was strongly accumulated within s-IBM muscle-fiber aggregates, where it closely co-localized with p62, ubiquitin, and phosphorylated tau; (b) immunoblots, was increased threefold (p < 0.001); and (c) immunoprecipitation, was associated with p62 and LC3. By real-time PCR, NBR1 mRNA was increased twofold (p < 0.01). None of the various disease- and normal-control muscle biopsies had any NBR1 abnormality. In cultured human muscle fibers, NBR1 also physically associated with both p62 and LC3, and experimental inhibition of either the 26S proteasome or the lysosomal activity resulted in NBR1 increase. Our demonstration of NBR1 abnormalities in s-IBM provides further evidence that altered protein degradation pathways may be critically involved in the s-IBM pathogenesis. Accordingly, attempts to unblock defective protein degradation might be a therapeutic strategy for s-IBM patients.     

Effects of tail suspension on serum testosterone and molecular targets regulating muscle mass

Introduction: The contribution of reduced testosterone levels to tail suspension (TS)‐induced muscle atrophy remains equivocal. The molecular mechanism by which testosterone regulates muscle mass during TS has not been investigated. Methods: Effects of TS on serum testosterone levels, muscle mass, and expression of muscle atrophy‐ and hypertrophy‐inducing targets were measured in soleus (SOL) and extensor digitorum longus (EDL) muscles after testosterone administration during 1, 5, and 14 days of TS in male mice. Results: TS produced an increase followed by a transient drop in testosterone levels. Muscle atrophy was associated with downregulation of Igf1 and upregulation of Mstn, Redd1, Atrogin‐1, and MuRF1 mRNA with clear differences in Igf1, Mstn, and MAFbx/Atrogin‐1 gene expression between SOL and EDL. Testosterone supplementation did not affect muscle mass or protein expression levels during TS. Conclusions The known anabolic effects of testosterone are not sufficient to ameliorate loss of muscle mass during TS. Muscle Nerve 52 : 278–288, 2015     

Needle electromyography and histopathologic correlation in myopathies

Introduction: The molecular mechanism of immune-mediated necrotizing myopathy (IMNM) remains unknown. Autophagy impairment, described in autoimmune diseases, is a key process in myofiber protein degradation flux and muscle integrity and has not been studied in IMNM. Methods: Muscle biopsies from patients with IMNM (n = 40), dermatomyositis (DM; 24), polymyositis (PM; 8), polymyositis with mitochondrial pathology (4), sporadic inclusion body myositis (8), and controls (6) were compared by immunohistochemistry. Results: The proportions of myofibers containing autophagy markers LC3b and p62 were higher in IMNM than in DM or PM and correlated with creatine kinase levels. In IMNM, compartmentalized LC3b puncta were located in regenerating and degenerating myofibers surrounded by major histocompatibility complex type II⁺ inflammatory cells. Several IMNM myofibers accumulated ubiquitin and misfolded protein. Discussion: The detection of LC3b⁺ or p62⁺ myofibers could be used in differentiating IMNM from PM. The identification of autophagy-modifying molecules potentially could improve patients’ outcomes. Muscle Nerve, 2019     

Paraplegia increases skeletal muscle autophagy

Introduction: Paraplegia results in significant skeletal muscle atrophy through increases in skeletal muscle protein breakdown. Recent work has identified a novel SIRT1–p53 pathway that is capable of regulating autophagy and protein breakdown. Methods: Soleus muscle was collected from 6 male Sprague‐Dawley rats 10 weeks after complete T4–5 spinal cord transection (paraplegia group) and 6 male sham‐operated rats (control group). We utilized immunoblotting methods to measure intracellular proteins and quantitative real‐time polymerase chain reaction to measure the expression of skeletal muscle microRNAs. Results: SIRT1 protein expression was 37% lower, and p53 acetylation (LYS379) was increased in the paraplegic rats (P < 0.05). Atg7 and Beclin‐1, markers of autophagy induction, were elevated in the paraplegia group compared with controls (P < 0.05). Conclusions: Severe muscle atrophy resulting from chronic paraplegia appears to increase skeletal muscle autophagy independent of SIRT1 signaling. We conclude that chronic paraplegia may cause an increase in autophagic cell death and negatively impact skeletal muscle protein balance. Muscle Nerve 46: 793–798, 2012     

Impaired autophagy correlates with golden retriever muscular dystrophy phenotype

Introduction: Duchenne muscular dystrophy (DMD) and golden retriever muscular dystrophy (GRMD) are X‐linked disorders caused by mutations in the DMD gene. Autophagy was recently identified as a secondary therapeutic target for DMD. We hypothesized that autophagy would be reduced in GRMD. Methods: Autophagic gene and protein expression was assessed in normal and GRMD skeletal muscles and correlated with phenotypic biomarkers. Results: Muscles were differentially affected. Autophagy gene levels were lower than normal in the GRMD cranial sartorius (CS) but similar in the vastus lateralis (VL). Protein markers of autophagic flux, LC3B‐II and p62, were higher in both GRMD muscles, in keeping with impaired autophagy. Protein levels correlated with a more severe phenotype. Autophagic structures were found in necrotic, fast‐twitch GRMD myofibers. Discussion: Our data suggest that autophagy is impaired in certain GRMD muscles. Differential GRMD CS involvement emphasizes that therapeutic modulation of autophagy could require specific muscle targeting. Muscle Nerve 58 : 418–426, 2018     

Involvement of Macroautophagy in Multiple System Atrophy and Protein Aggregate Formation in Oligodendrocytes

α-Synuclein-containing glial cytoplasmic inclusions (GCIs) originating in oligodendrocytes are the characteristic hallmark for neuropathological diagnosis of multiple system atrophy (MSA). α-Synuclein can be degraded either by the proteasomal machinery or by autophagy, a lysosomal pathway which involves the formation of autophagosomes. The autophagosome takes up polyubiquitinated proteins via the autophagosomal protein LC3 and the ubiquitin binding protein p62. In the present study, neuropathological examination of seven MSA cases revealed that LC3-immunoreactivity is found to be associated with α-synuclein-positive GCIs. These are also prominently stained by antibodies against p62 and ubiquitin, indicating that the autophagic pathway is upregulated during pathogenesis, which might be due to a persistent downregulation of proteasomal activity. To further address this question in a cellular context, we have investigated whether proteasomal inhibition in cultured rat brain oligodendrocytes promotes the recruitment of LC3 and p62 to protein aggregates. The data show that the autophagic marker LC3-II is upregulated and LC3 is recruited to the growing protein aggregates in cultured oligodendrocytes when the proteasome is impaired. However, aggregated proteins remain in the oligodendroglial cytoplasm and cannot be cleared efficiently. In conclusion, autophagy and the ubiquitin proteasome system are closely connected, and the presence of LC3-positive vesicles in GCIs indicates that macroautophagy participates in MSA pathogenesis.     

Apigenin inhibits sciatic nerve denervation–induced muscle atrophy

Introduction: Apigenin (AP) has been reported to elicit anti‐inflammatory effects. In this study, we investigated the effect of AP on sciatic nerve denervation–induced muscle atrophy. Methods: Sciatic nerve–denervated mice were fed a 0.1% AP‐containing diet for 2 weeks. Muscle weight and cross‐sectional area (CSA), and the expression of atrophic genes and inflammatory cytokines in the gastrocnemius were analyzed. Results: Denervation significantly induced muscle atrophy. However, values for muscle weight and CSA were greater in the denervated muscle of the AP mice than the controls. AP suppressed the expression of MuRF1, but upregulated both myosin heavy chain (MHC) and MHC type IIb. AP also significantly suppressed expression of tumor necrosis‐alpha in the gastrocnemius and soleus muscles, and interleukin‐6 expression in the soleus muscle. Discussion: AP appears to inhibit denervation‐induced muscle atrophy, which may be due in part to its inhibitory effect on inflammatory processes within muscle. Muscle Nerve 58 : 314–318, 2018     

Ultrastructural changes in LGMD1F

A large Italo‐Spanish kindred with autosomal‐dominant inheritance has been reported with proximal limb and axial muscle weakness. Clinical, histological and genetic features have been described. A limb girdle muscular dystrophy 1F (LGMD1F) disease locus at chromosome 7q32.1–32.2 has been previously identified. We report a muscle pathological study of two patients (mother and daughter) from this family. Muscle morphologic findings showed increased fiber size variability, fiber atrophy, and acid‐phosphatase‐positive vacuoles. Immunofluorescence against desmin, myotilin, p62 and LC3 showed accumulation of myofibrils, ubiquitin binding protein aggregates and autophagosomes. The ultrastructural study confirmed autophagosomal vacuoles. Many alterations of myofibrillar component were detected, such as prominent disarray, rod‐like structures with granular aspect, and occasionally, cytoplasmic bodies. Our ultrastructural data and muscle pathological features are peculiar to LGMD1F and support the hypothesis that the genetic defect leads to a myopathy phenotype associated with disarrangement of the cytoskeletal network.     

Ubiquitin‐negative,eosinophilic neuronal cytoplasmic inclusions associated with stress granules and autophagy: An immunohistochemical investigation of two cases

Identification of the proteinaceous components of the pathological inclusions is an important step in understanding the associated disease mechanisms. We immunohistochemically examined two previously reported cases with eosinophilic neuronal cytoplasmic inclusions (NCIs) (case 1, Mori et al. Neuropathology 2010; 30: 648–53; case 2, Kojima et al. Acta Pathol Jpn 1990; 40: 785–91) using 67 antibodies against proteins related to cytoskeletal constituents, ubiquitin‐proteasome system, autophagy‐lysosome pathway and stress granule formation. Regional distribution pattern of eosinophilic NCIs in case 1 was substantially different from that in case 2. However, NCIs in both cases were immunonegative for ubiquitin and p62 and were immunopositive for stress granule markers as well as autophagy‐related proteins, including valosin‐containing protein. Considering that eukaryotic stress granules are cleared by autophagy and valosin‐containing protein function, our findings suggest that eosinophilic NCIs in the present two cases may represent the process of autophagic clearance of stress granules.     

Branched‐chain amino acids protect against dexamethasone‐induced soleus muscle atrophy in rats

We investigated the utility of branched‐chain amino acids (BCAA) in dexamethasone‐induced muscle atrophy. Dexamethasone (600 μg/kg, intraperitoneally) and/or BCAA (600 mg/kg, orally) were administered for 5 days in rats, and the effect of BCAA on dexamethasone‐induced muscle atrophy was evaluated. Dexamethasone decreased total protein concentration of rat soleus muscles. Concomitant administration of BCAA reversed the decrease. Dexamethasone decreased mean cross‐sectional area of soleus muscle fibers, which was reversed by BCAA. Dexamethasone increased atrogin‐1 expression, which has been reported to play a pivotal role in muscle atrophy. The increased expression of atrogin‐1 mRNA was significantly attenuated by BCAA. Furthermore, dexamethasone‐induced conversion from microtubule‐associated protein 1 light chain 3 (LC3)‐I to LC3‐II, which is an indicator of autophagy, was blocked by BCAA. These findings suggest that BCAA decreased protein breakdown to prevent muscle atrophy. BCAA administration appears to be useful for prevention of steroid myopathy. Muscle Nerve, 2010     

Pain and small fiber function in charcot–marie–tooth disease type 1A

Introduction: Charcot–Marie–Tooth (CMT) disease type 1A is the most common form of CMT. The main clinical features are distal weakness, sensory loss, and skeletal deformities. Although pain is a frequent complaint, small fiber involvement in CMT1A has not been studied extensively. Methods: We assessed pain and small fiber involvement in 49 CMT1A patients using a variety of pain scales, pain questionnaires, and thermal thresholds. Results: Forty‐three of 49 patients (88%) complained of pain. The pain was localized to the feet in 61% of patients. Only 18% of patients had neuropathic pain. Cold and warm detection thresholds were elevated in 53% and 12% of patients, respectively. Conclusions: Our findings confirm that CMT1A patients have significant pain, which is more likely to be multifactorial in origin and suggests that a proportion of patients have small fiber dysfunction affecting mainly thinly myelinated Aδ fibers. Muscle Nerve 50 : 366–371, 2014