Muscle RING-finger Protein-1 Research Articles

Plasma from septic shock patients induces loss of muscle protein

IntroductionICU-acquired muscle weakness commonly occurs in patients with septic shock and is associated with poor outcome. Although atrophy is known to be involved, it is unclear whether ligands in plasma from these patients are responsible for initiating degradation of muscle proteins. The aim of the present study was to investigate if plasma from septic shock patients induces skeletal muscle atrophy and to examine the time course of plasma-induced muscle atrophy during ICU stay.MethodsPlasma was derived from septic shock patients within 24 hours after hospital admission (n = 21) and healthy controls (n = 12). From nine patients with septic shock plasma was additionally derived at two, five and seven days after ICU admission. These plasma samples were added to skeletal myotubes, cultured from murine myoblasts. After incubation for 24 hours, myotubes were harvested and analyzed on myosin content, mRNA expression of E3-ligase and Nuclear Factor Kappa B (NFκB) activity. Plasma samples were analyzed on cytokine concentrations.ResultsMyosin content was approximately 25% lower in myotubes exposed to plasma from septic shock patients than in myotubes exposed to plasma from controls (P < 0.01). Furthermore, patient plasma increased expression of E3-ligases Muscle RING Finger protein-1 (MuRF-1) and Muscle Atrophy F-box protein (MAFbx) (P < 0.01), enhanced NFκB activity (P < 0.05) and elevated levels of ubiquitinated myosin in myotubes. Myosin loss was significantly associated with elevated plasma levels of interleukin (IL)-6 in septic shock patients (P < 0.001). Addition of antiIL-6 to septic shock plasma diminished the loss of myosin in exposed myotubes by approximately 25% (P < 0.05). Patient plasma obtained later during ICU stay did not significantly reduce myosin content compared to controls.ConclusionsPlasma from patients with septic shock induces loss of myosin and activates key regulators of proteolysis in skeletal myotubes. IL-6 is an important player in sepsis-induced muscle atrophy in this model. The potential to induce atrophy is strongest in plasma obtained during the early phase of human sepsis.

Procysteine Increases Alcohol-depleted Glutathione Stores in Rat Plantaris Following a Period of Abstinence

Aims: To assess the effectiveness of procysteine (PRO) supplementation provided during a period of abstinence (ABS) on alcohol-induced skeletal muscle atrophy and oxidant stress. Methods: Age- and gender-matched Sprague–Dawley rats were fed the Lieber–DeCarli liquid diet containing either alcohol or an isocaloric substitution (control diet) for 12 week. Next, subgroups of alcohol-fed rats were fed the control diet for 2 week (ABS) supplemented with either PRO (0.35%, w/v) or vehicle. Plantaris morphology was assessed by hematoxylin and eosin staining. Total, reduced and oxidized glutathione (GSH) levels and total antioxidant potential were determined by commercially available assay kits. Antibody arrays were used to determine cytokine levels. Real-time polymerase chain reaction was used to determine gene expressions of two E3 ubiquitin ligases, atrogin-1 and muscle ring finger protein-1 (MuRF-1). Results: Plantaris muscles from alcohol-fed rats displayed extensive atrophy, as well as decreased GSH levels, a trend for decreased total antioxidant potential and elevated atrogin-1 and MuRF-1 mRNA levels. GSH levels and total antioxidant potential continued to decrease during 2 weeks of ABS from alcohol, which were normalized in abstinent rats provided PRO. Gene levels of both E3 ligases returned to baseline during ABS. In parallel, plantaris cross-sectional area increased in both groups during ABS. Conclusions: PRO supplementation during ABS significantly attenuated alcohol-induced redox stress compared with untreated abstinent rats. Thus, our data may suggest that GSH restoration therapy may provide therapeutic benefits to the overall antioxidant state of skeletal muscle when prescribed in conjunction with an established detoxification program for recovering alcoholics.

Interactions with M-band Titin and Calpain 3 Link Myospryn (CMYA5) to Tibial and Limb-girdle Muscular Dystrophies

Mutations in the C terminus of titin, situated at the M-band of the striated muscle sarcomere, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy (LGMD) type 2J. Mutations in the protease calpain 3 (CAPN3), in turn, lead to LGMD2A, and secondary CAPN3 deficiency in LGMD2J suggests that the pathomechanisms of the diseases are linked. Yeast two-hybrid screens carried out to elucidate the molecular pathways of TMD/LGMD2J and LGMD2A resulted in the identification of myospryn (CMYA5, cardiomyopathy-associated 5) as a binding partner for both M-band titin and CAPN3. Additional yeast two-hybrid and coimmunoprecipitation studies confirmed both interactions. The interaction of myospryn and M-band titin was supported by localization of endogenous and transfected myospryn at the M-band level. Coexpression studies showed that myospryn is a proteolytic substrate for CAPN3 and suggested that myospryn may protect CAPN3 from autolysis. Myospryn is a muscle-specific protein of the tripartite motif superfamily, reported to function in vesicular trafficking and protein kinase A signaling and implicated in the pathogenesis of Duchenne muscular dystrophy. The novel interactions indicate a role for myospryn in the sarcomeric M-band and may be relevant for the molecular pathomechanisms of TMD/LGMD2J and LGMD2A.

Mechanism of Attenuation of Skeletal Muscle Atrophy by Zinc-α2-Glycoprotein

The mechanism by which the adipokine zinc-α2-glycoprotein (ZAG) increases the mass of gastrocnemius, but not soleus muscle of diabetic mice, has been evaluated both in vivo and in vitro. There was an increased phosphorylation of both double-stranded RNA-dependent protein kinase and its substrate, eukaryotic initiation factor-2α, which was attenuated by about two-thirds in gastrocnemius but not soleus muscle of ob/ob mice treated with ZAG (50 μg, iv daily) for 5 d. ZAG also reduced the expression of the phospho forms of p38MAPK and phospholipase A2, as well as expression of the ubiquitin ligases (E3) muscle atrophy F-box/atrogin-1 and muscle RING finger protein, and the increased activity of both caspase-3 and casapse-8 to values found in nonobese controls. ZAG also increased the levels of phospho serine-threonine kinase and mammalian target of rapamycin in gastrocnemius muscle and reduced the phosphorylation of insulin receptor substrate-1 (Ser307) associated with insulin resistance. Similar changes were seen with ZAG when murine myotubes were incubated with high glucose concentrations (10 and 25 mm), showing that the effect of ZAG was direct. ZAG produced an increase in cAMP in murine myotubes, and the effects of ZAG on protein synthesis and degradation in vitro could be replicated by dibutyryl cAMP. ZAG increased cAMP levels of gastrocnemius but not soleus muscle. These results suggest that protein accretion in skeletal muscle in response to ZAG may be due to changes in intracellular cAMP and also that ZAG may have a therapeutic application in the treatment of muscle wasting conditions.

Activation of AMPK inhibits cardiomyocyte hypertrophy by modulating of the FOXO1/MuRF1 signaling pathway in vitro

To examine the inhibitory effects of adenosine monophosphate-activated protein kinase (AMPK) activation on cardiac hypertrophy in vitro and to investigate the underlying molecular mechanisms. Cultured neonatal rat cardiomyocytes were treated with the specific AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the specific AMPK antagonist Compound C, and then stimulated with phenylephrine (PE). The Muscle RING finger 1 (MuRF1)-small interfering RNA (siRNA) was transfected into cardiomyocytes using Lipofectamine 2000. The surface area of cultured cardiomyocytes was measured using planimetry. The protein degradation was determined using high performance liquid chromatography (HPLC). The expression of beta-myosin heavy chain (beta-MHC) and MuRF1, as well as the phosphorylation levels of AMPK and Forkhead box O 1 (FOXO1), were separately measured using Western blot or real-time polymerase chain reaction. Activation of AMPK by AICAR 0.5 mmol/L inhibited PE-induced increase in cardiomyocyte area and beta-MHC protein expression and PE-induced decrease in protein degradation. Furthermore, AMPK activation increased the activity of transcription factor FOXO1 and up-regulated downstream atrogene MuRF1 mRNA and protein expression. Treatment of hypertrophied cardiomyocytes with Compound C 1 micromol/L blunted the effects of AMPK on cardiomyocyte hypertrophy and changes to the FOXO1/MuRF1 pathway. The effects of AICAR on cardiomyocyte hypertrophy were also blocked after MuRF1 was silenced by transfection of cardiomyocytes with MuRF1-siRNA. The present study demonstrates that AMPK activation attenuates cardiomyocyte hypertrophy by modulating the atrophy-related FOXO1/MuRF1 signaling pathway in vitro.

Effect of pulmonary rehabilitation on muscle remodelling in cachectic patients with COPD

It is known that non-cachectic patients with chronic obstructive pulmonary disease (COPD) respond well to pulmonary rehabilitation, but whether cachectic COPD patients are capable of adaptive responses is both important and unknown. 10 cachectic and 19 non-cachectic COPD patients undertook high-intensity cycling training, at the same relative intensity, for 45 min x day(-1), 3 days x week(-1) for 10 weeks. Before and after rehabilitation vastus lateralis muscle biopsies were analysed morphologically and for the expression of muscle remodelling factors (insulin-like growth factor (IGF)-I, myogenic differentiation factor D (MyoD), tumour necrosis factor (TNF)-alpha, nuclear factor (NF)-kappaB and myostatin) and key components of ubiquitin-mediated proteolytic systems (muscle ring finger protein (MURF)-1 and Atrogin-1). Rehabilitation improved peak work-rate and the 6-min walk distance similarly in non-cachectic (18+/-3% and 42+/-13 m, respectively) and cachectic (16+/-2% and 53+/-16 m, respectively) patients, but quality of life only improved in non-cachectic COPD. Mean muscle fibre cross-sectional area increased in both groups, but significantly less in cachectic (7+/-2%) than in non-cachectic (11+/-2%) patients. Both groups equally decreased the proportion of type IIb fibres and increased muscle capillary/fibre ratio. IGF-I mRNA expression increased in both groups, but IGF-I protein levels increased more in non-cachectic COPD. MyoD was upregulated, whereas myostatin was downregulated at the mRNA and protein level only in non-cachectic patients. Whilst rehabilitation had no effect on TNF-alpha expression, it decreased the activation of the transcription factor NF-kappaB in both groups by the same amount. Atrogin-1 and MURF-1 expression were increased in cachectic COPD, but it was decreased in non-cachectic patients. Cachectic COPD patients partially retain the capacity for peripheral muscle remodelling in response to rehabilitation and are able to increase exercise capacity as much as those without cachexia, even if they exhibit both quantitative and qualitative differences in the type of muscle fibre remodelling in response to exercise training.

P38 MAPK links oxidative stress to autophagy-related gene expression in cachectic muscle wasting

Oxidative stress is a primary trigger of cachectic muscle wasting, but the signaling pathway(s) that links it to the muscle wasting processes remains to be defined. Here, we report that activation of p38 mitogen-activated protein kinase (MAPK) (phosphorylation) and increased oxidative stress (trans-4-hydroxy-2-nonenal protein modification) in skeletal muscle occur as early as 8 h after lipopolysaccharide (1 mg/kg) and 24 h after dexamethasone (25 mg/kg) injection (intraperitoneal) in mice, concurrent with upregulation of autophagy-related genes, Atg6, Atg7, and Atg12. Treating cultured C2C12 myotubes with oxidant hydrogen peroxide (4 h) resulted in increased p38 phosphorylation and reduced FoxO3 phosphorylation along with induced Atg7 mRNA expression without activation of NF-kappaB or FoxO3a transcriptional activities. Furthermore, inhibition of p38alpha/beta by SB202190 blocked hydrogen peroxide-induced atrophy with diminished upregulation of Atg7 and atrogenes [muscle atrophy F-box protein (MAFbx/Atrogin-1), muscle ring finger protein 1 (MuRF-1), and Nedd4]. These findings provide direct evidence for p38alpha/beta MAPK in mediating oxidative stress-induced autophagy-related genes, suggesting that p38alpha/beta MAPK regulates both the ubiquitin-proteasome and the autophagy-lysosome systems in muscle wasting.

Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms

Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C. Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by >60%, but also the level of myosin heavy chains (MHCs) by >40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels. These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.

Procysteine Stimulates Expression of Key Anabolic Factors and Reduces Plantaris Atrophy in Alcohol‐Fed Rats

Long-term alcohol ingestion may produce severe oxidant stress and lead to skeletal muscle dysfunction. Emerging evidence has suggested that members of the interleukin-6 (IL-6) family of cytokines play diverse roles in the regulation of skeletal muscle mass. Thus, our goals were (i) to minimize the degree of oxidant stress and attenuate atrophy by supplementing the diets of alcohol-fed rats with the glutathione precursor, procysteine, and (ii) to identify the roles of IL-6 family members in alcoholic myopathy. Age- and gender-matched Sprague-Dawley rats were fed the Lieber-DeCarli liquid diet containing either alcohol or an isocaloric substitution (control diet) for 35 weeks. Subgroups of alcohol-fed rats received procysteine (0.35%, w/v) for the final 12 weeks. Plantaris morphology was assessed by hematoxylin and eosin staining. Major components of glutathione metabolism were determined using assay kits. Real-time PCR was used to determine expression levels of several genes. Plantaris muscles from alcohol-fed rats displayed extensive atrophy, as well as decreased glutathione levels, decreased activities of glutathione reductase and glutathione peroxidase, decreased superoxide dismutase (SOD)-2 (Mn-SOD2), and increased NADPH oxidase-1 gene expression-each indicative of significant oxidant stress. Alcohol also induced gene expression of catabolic factors including IL-6, oncostatin M, atrogin-1, muscle ring finger protein-1, and IGFBP-1. Procysteine treatment attenuated plantaris atrophy, restored glutathione levels, and increased catalase, Cu/Zn-SOD1, and Mn-SOD2 mRNA expression, but did not reduce other markers of oxidant stress or levels of these catabolic factors. Instead, procysteine stimulated gene expression of anabolic factors such as insulin-like growth factor-1, ciliary neurotrophic factor, and cardiotrophin-1. Procysteine significantly attenuated, but did not completely abrogate, alcohol-induced oxidant stress or catabolic factors. Rather, procysteine minimized the extent of plantaris atrophy by inducing components of several anabolic pathways. Therefore, anti-oxidant treatments such as procysteine supplementation may benefit individuals with alcoholic myopathy.

Smad2 and 3 transcription factors control muscle mass in adulthood

Loss of muscle mass occurs in a variety of diseases, including cancer, chronic heart failure, aquired immunodeficiency syndrome, diabetes, and renal failure, often aggravating pathological progression. Preventing muscle wasting by promoting muscle growth has been proposed as a possible therapeutic approach. Myostatin is an important negative modulator of muscle growth during myogenesis, and myostatin inhibitors are attractive drug targets. However, the role of the myostatin pathway in adulthood and the transcription factors involved in the signaling are unclear. Moreover, recent results confirm that other transforming growth factor-beta (TGF-beta) members control muscle mass. Using genetic tools, we perturbed this pathway in adult myofibers, in vivo, to characterize the downstream targets and their ability to control muscle mass. Smad2 and Smad3 are the transcription factors downstream of myostatin/TGF-beta and induce an atrophy program that is muscle RING-finger protein 1 (MuRF1) independent. Furthermore, Smad2/3 inhibition promotes muscle hypertrophy independent of satellite cells but partially dependent of mammalian target of rapamycin (mTOR) signaling. Thus myostatin and Akt pathways cross-talk at different levels. These findings point to myostatin inhibitors as good drugs to promote muscle growth during rehabilitation, especially when they are combined with IGF-1-Akt activators.

Protein synthesis and the expression of growth-related genes are altered by running in human vastus lateralis and soleus muscles

Recent evidence suggests aerobic exercise may help preserve soleus muscle mass during unloading. The purpose of this investigation was to examine the muscle-specific metabolic response to running as it relates to muscle growth. Mixed-muscle protein synthesis [fractional synthetic rate (FSR)] and gene expression (GE) were examined in the vastus lateralis (VL) and soleus (SOL) muscles from eight men (26 +/- 2 yr; Vo(2max) 63 +/- 2 ml.kg(-1).min(-1)) before and after a 45-min level-grade treadmill run at 77 +/- 1% intensity. Muscle glycogen utilization was similar between muscles. Resting FSR was similar between the VL (0.080 +/- 0.007 %/h) and SOL (0.086 +/- 0.008 %/h) and was higher (P < 0.05) 24 h postexercise compared with rest for both muscles. The absolute change in FSR was not different between muscles (0.030 +/- 0.007 vs. 0.037 +/- 0.012 %/h for VL and SOL). At baseline, myostatin GE was approximately twofold higher (P < 0.05) in SOL compared with VL, while no other muscle-specific differences in GE were present. After running, myostatin GE was suppressed (P < 0.05) in both muscles at 4 h and was higher (P < 0.05) than baseline at 24 h for VL only. Muscle regulatory factor 4 mRNA was elevated (P < 0.05) at 4 h in both SOL and VL; MyoD and peroxisome-proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) were higher (P < 0.05) at 4 h, and forkhead box [FOXO]3A was higher at 24 h in SOL only, while muscle-RING-finger protein-1 (MuRF-1) was higher (P < 0.05) at 4 h in VL only. Myogenin and atrogin-1 GE were unaltered. The similar increases between muscles in FSR support running as part of the exercise countermeasure to preserve soleus mass during unloading. The subtle differences in GE suggest a potential mechanism for muscle-specific adaptations to chronic run training.

Effect of HIV-1-related protein expression on cardiac and skeletal muscles from transgenic rats

BackgroundHuman immunodeficiency virus type 1 (HIV-1) infection and the consequent acquired immunodeficiency syndrome (AIDS) has protean manifestations, including muscle wasting and cardiomyopathy, which contribute to its high morbidity. The pathogenesis of these myopathies remains partially understood, and may include nutritional deficiencies, biochemical abnormalities, inflammation, and other mechanisms due to viral infection and replication. Growing evidence has suggested that HIV-1-related proteins expressed by the host in response to viral infection, including Tat and gp120, may also be involved in the pathophysiology of AIDS, particularly in cells or tissues that are not directly infected with HIV-1. To explore the potentially independent effects of HIV-1-related proteins on heart and skeletal muscles, we used a transgenic rat model that expresses several HIV-1-related proteins (e.g., Tat, gp120, and Nef). Outcome measures included basic heart and skeletal muscle morphology, glutathione metabolism and oxidative stress, and gene expressions of atrogin-1, muscle ring finger protein-1 (MuRF-1) and Transforming Growth Factor-β1 (TGFβ1), three factors associated with muscle catabolism.ResultsConsistent with HIV-1 associated myopathies in humans, HIV-1 transgenic rats had increased relative heart masses, decreased relative masses of soleus, plantaris and gastrocnemius muscles, and decreased total and myosin heavy chain type-specific plantaris muscle fiber areas. In both tissues, the levels of cystine (Cyss), the oxidized form of the anti-oxidant cysteine (Cys), and Cyss:Cys ratios were significantly elevated, and cardiac tissue from HIV-1 transgenic rats had altered glutathione metabolism, all reflective of significant oxidative stress. In HIV-1 transgenic rat hearts, MuRF-1 gene expression was increased. Further, HIV-1-related protein expression also increased atrogin-1 (~14- and ~3-fold) and TGFβ1 (~5-fold and ~3-fold) in heart and plantaris muscle tissues, respectively.ConclusionWe provide compelling experimental evidence that HIV-1-related proteins can lead to significant cardiac and skeletal muscle complications independently of viral infection or replication. Our data support the concept that HIV-1-related proteins are not merely disease markers, but rather have significant biological activity that may lead to increased oxidative stress, the stimulation of redox-sensitive pathways, and altered muscle morphologies. If correct, this pathophysiological scheme suggests that the use of dietary thiol supplements could reduce skeletal and cardiac muscle dysfunction in HIV-1-infected individuals.

Muscle RING-Finger Protein-1 (MuRF1) as a Connector of Muscle Energy Metabolism and Protein Synthesis

During pathophysiological muscle wasting, a family of ubiquitin ligases, including muscle RING-finger protein-1 (MuRF1), has been proposed to trigger muscle protein degradation via ubiquitination. Here, we characterized skeletal muscles from wild-type (WT) and MuRF1 knockout (KO) mice under amino acid (AA) deprivation as a model for physiological protein degradation, where skeletal muscles altruistically waste themselves to provide AAs to other organs. When WT and MuRF1 KO mice were fed a diet lacking AA, MuRF1 KO mice were less susceptible to muscle wasting, for both myocardium and skeletal muscles. Under AA depletion, WT mice had reduced muscle protein synthesis, while MuRF1 KO mice maintained nonphysiologically elevated levels of skeletal muscle protein de novo synthesis. Consistent with a role of MuRF1 for muscle protein turnover during starvation, the concentrations of essential AAs, especially branched-chain AAs, in the blood plasma significantly decreased in MuRF1 KO mice under AA deprivation. To clarify the molecular roles of MuRF1 for muscle metabolism during wasting, we searched for MuRF1-associated proteins using pull-down assays and mass spectrometry. Muscle-type creatine kinase (M-CK), an essential enzyme for energy metabolism, was identified among the interacting proteins. Coexpression studies revealed that M-CK interacts with the central regions of MuRF1 including its B-box domain and that MuRF1 ubiquitinates M-CK, which triggers the degradation of M-CK via proteasomes. Consistent with MuRF1's role of adjusting CK activities in skeletal muscles by regulating its turnover in vivo, we found that CK levels were significantly higher in the MuRF1 KO mice than in WT mice. Glucocorticoid modulatory element binding protein-1 and 3-hydroxyisobutyrate dehydrogenase, previously identified as potential MuRF1-interacting proteins, were also ubiquitinated MuRF1-dependently. Taken together, these data suggest that, in a multifaceted manner, MuRF1 participates in the regulation of AA metabolism, including the control of free AAs and their supply to other organs under catabolic conditions, and in the regulation of ATP synthesis under metabolic-stress conditions where MuRF1 expression is induced.

The E3 Ligase MuRF1 Degrades Myosin Heavy Chain Protein in Dexamethasone-Treated Skeletal Muscle

Skeletal muscle atrophy occurs as a side effect of treatment with synthetic glucocorticoids such as dexamethasone (DEX) and is a hallmark of cachectic syndromes associated with increased cortisol levels. The E3 ubiquitin ligase MuRF1 (muscle RING finger protein 1) is transcriptionally upregulated by DEX treatment. Differentiated myotubes treated with DEX undergo depletion of myosin heavy chain protein (MYH), which physically associates with MuRF1. This loss of MYH can be blocked by inhibition of MuRF1 expression. When wild-type and MuRF1(-/-) mice are treated with DEX, the MuRF1(-/-) animals exhibit a relative sparing of MYH. In vitro, MuRF1 is shown to function as an E3 ubiquitin ligase for MYH. These data identify the mechanism by which MYH is depleted under atrophy conditions and demonstrate that inhibition of a single E3 ligase, MuRF1, is sufficient to maintain this important sarcomeric protein.

Expression patterns of atrogenic and ubiquitin proteasome component genes with exercise: effect of different loading patterns and repeated exercise bouts

Unaccustomed exercise is known to produce strength loss, soreness, and myocellular disruption. With repeated application of exercise stimuli, the appearance of these indexes of muscle damage is attenuated, the so-called "repeated bout effect." No direct connection has been established between this repeated bout effect and exercise-induced increases in protein turnover, but it appears that a degree of tolerance is developed toward exercise for both. The present study sought to investigate markers of protein degradation by determining the expression of components related to the ubiquitin-proteasome system (UPS) with repeated exercise bouts. Healthy men carried out 30 min of bench stepping, performing eccentric work with one and concentric work with the other leg (n = 14), performing a duplicate exercise bout 8 wk later. A nonexercising control group was included (n = 6). RNA was extracted from muscle biopsies representing time points preexercise, +3 h, +24 h, and +7 days, and selected mRNA species were quantified using Northern blotting. The exercise model proved sufficient to produce a repeated bout effect in terms of strength and soreness. For forkhead box O transcription factor 1 (FOXO1) and muscle RING finger protein-1 (MURF1), strong upregulations were seen exclusively with concentric loading (P < 0.001), while atrogin-1 displayed a strong downregulation exclusively in response to eccentric exercise (P < 0.001). For MURF1 transcription, the first bout produced a downregulation that persisted until the second bout (P < 0.01). In conclusion, the UPS is modulated differentially in response to varying loading modalities and with different time frames in a way that to some extent reflects changes in protein metabolism known to take place with exercise.

Muscle Atrophy and Hypertrophy Signaling in Patients with Chronic Obstructive Pulmonary Disease

The molecular mechanisms of muscle atrophy in chronic obstructive pulmonary disease (COPD) are poorly understood. In wasted animals, muscle mass is regulated by several AKT-related signaling pathways. To measure the protein expression of AKT, forkhead box class O (FoxO)-1 and -3, atrogin-1, the phosphophrylated form of AKT, p70(S6K) glycogen synthase kinase-3beta (GSK-3beta), eukaryotic translation initiation factor 4E binding protein-1 (4E-BP1), and the mRNA expression of atrogin-1, muscle ring finger (MuRF) protein 1, and FoxO-1 and -3 in the quadriceps of 12 patients with COPD with muscle atrophy and 10 healthy control subjects. Five patients with COPD with preserved muscle mass were subsequently recruited and were compared with six patients with low muscle mass. Protein contents and mRNA expression were measured by Western blot and quantitative polymerase chain reaction, respectively. The levels of atrogin-1 and MuRF1 mRNA, and of phosphorylated AKT and 4E-BP1 and FoxO-1 proteins, were increased in patients with COPD with muscle atrophy compared with healthy control subjects, whereas atrogin-1, p70(S6K), GSK-3beta, and FoxO-3 protein levels were similar. Patients with COPD with muscle atrophy showed an increased expression of p70(S6K), GSK-3beta, and 4E-BP1 compared with patients with COPD with preserved muscle mass. An increase in atrogin-1 and MuRF1 mRNA and FoxO-1 protein content was observed in the quadriceps of patients with COPD. The transcriptional regulation of atrogin-1 and MuRF1 may occur via FoxO-1, but independently of AKT. The overexpression of the muscle hypertrophic signaling pathways found in patients with COPD with muscle atrophy could represent an attempt to restore muscle mass.

The Generation of the Oxidized Form of Creatine Kinase Is a Negative Regulation on Muscle Creatine Kinase

Muscle creatine kinase (CK) is a crucial enzyme in energy metabolism, and it exists in two forms, the reduced form (R-CK) and the oxidized form (O-CK). In contrast with R-CK, O-CK contained an intrachain disulfide bond in each subunit. Here we explored the properties of O-CK and its regulatory role on muscle CK. The intrachain disulfide bond in O-CK was demonstrated to be formed between Cys(74) and Cys(146) by site-directed mutagenesis. Biophysical analysis indicated that O-CK showed decreased catalytic activity and that it might be structurally unstable. Further assays through guanidine hydrochloride denaturation and proteolysis by trypsin and protease K revealed that the tertiary structure of O-CK was more easily disturbed than that of R-CK. Surprisingly, O-CK, unlike R-CK, cannot interact with the M-line protein myomesin through biosensor assay, indicating that O-CK might have no role in muscle contraction. Through in vitro ubiquitination assay, CK was demonstrated to be a specific substrate of muscle ring finger protein 1 (MURF-1). O-CK can be rapidly ubiquitinated by MURF-1, while R-CK can hardly be ubiquitinated, implying that CK might be degraded by the ATP-ubiquitin-proteasome pathway through the generation of O-CK. The results above were further confirmed by molecular modeling of the structure of O-CK. Therefore, it can be concluded that the generation of O-CK was a negative regulation of R-CK and that O-CK might play essential roles in the molecular turnover of MM-CK.

Muscle Ring Finger 1, but not Muscle Ring Finger 2, Regulates Cardiac Hypertrophy In Vivo

Muscle ring finger (MuRF) proteins have been implicated in transmitting mechanical forces to cell signaling pathways through their interactions with the giant protein titin. Recent evidence has linked mechanically-induced stimuli with the control of serum response factor activity and localization through MuRF2. This observation is particularly intriguing in the context of cardiac hypertrophy, where serum response factor transactivation is a key event necessary for the induction of cardiac hypertrophy in response to increased afterload. We have previously reported that MuRF1, which is also a titin-associated protein, exerts antihypertrophic activity in vitro. In the present study, we induced cardiac hypertrophy in mice lacking MuRF1 and MuRF2 to distinguish the physiologic role of these divergent proteins in vivo. We identified for the first time that MuRF1, but not MuRF2, plays a key role in regulating the induction of cardiac hypertrophy, likely by its direct interactions with serum response factor. These studies describe for the first time distinct and nonoverlapping functional characteristics of MuRF1 and MuRF2 in response to cardiac stress in vivo.

Rocuronium exacerbates mechanical ventilation–induced diaphragm dysfunction in rats

Nondepolarizing neuromuscular blocking agents are commonly used in the intensive care setting, but they have occasionally been associated with development of myopathy. In addition, diaphragmatic atrophy and a reduction in diaphragmatic force were reported after short-term controlled mechanical ventilation in animal models. We hypothesized that infusion of rocuronium, an aminosteroidal neuromuscular blocking agent, during 24 hrs of controlled mechanical ventilation would further alter diaphragm function and would enhance activation of the ubiquitin- proteasome pathway. Randomized, controlled experiment. Basic animal science laboratory. Male Wistar rats, 14 wks old. Rats were divided into four groups: a control group, a group of anesthetized rats breathing spontaneously for 24 hrs, and two groups submitted to mechanical ventilation for 24 hrs, receiving a continuous infusion of either 0.9% NaCl or rocuronium. In vitro diaphragm force was decreased more significantly after 24 hrs of mechanical ventilation combined with rocuronium infusion than after mechanical ventilation alone (e.g., tetanic force, -27%; p < .001 vs. mechanical ventilation). Similarly, the decrease in diaphragm type IIx/b fiber dimensions was more pronounced after mechanical ventilation with rocuronium treatment than with saline treatment (-38% and -29%, respectively; p < .001 vs. control). Diaphragm hydroperoxide levels increased similarly in both mechanically ventilated groups. Diaphragm muscle RING-finger protein-1 (MURF-1) messenger RNA expression, an E3 ligase of the ubiquitin-proteasome pathway, increased after mechanical ventilation (+212%, p < .001 vs. control) and increased further with combination of rocuronium (+320%, p < .001 vs. control). Significant correlations were found between expression of MURF-1 messenger RNA, diaphragm force, and type IIx/b fiber dimensions. Infusion of rocuronium during controlled mechanical ventilation leads to further deterioration of diaphragm function, additional atrophy of type IIx/b fibers, and an increase in MURF-1 messenger RNA in the diaphragm, which suggests an activation of the ubiquitin-proteasome pathway. These findings could be important with regard to weaning failure in patients receiving this drug for prolonged periods in the intensive care unit setting.

Atrophy-related ubiquitin ligases atrogin-1 and MuRF-1 are associated with uterine smooth muscle involution in the postpartum period

The regulation of cell size depends on a delicate balance between protein synthesis and breakdown. Skeletal and cardiac muscle adapt to hormonal and neuronal stimuli and can rapidly hypertrophy and atrophy; however, the extent to which these processes occur in smooth muscle is less clear. Atrophy in striated muscle results from enhanced protein breakdown and is associated with a common transcriptional profile and activation of the ubiquitin-proteasome pathway, including induction of the muscle-specific ubiquitin protein ligases atrogin-1 and muscle ring-finger protein 1 (MuRF-1). Here we show that atrogin-1 is also expressed in smooth muscle, and that both atrogin-1 and MuRF-1 are upregulated in the uterus following delivery, as rapid involution occurs. While these two genes are similarly induced in all types of muscle during rapid loss of cell mass, other striated muscle atrophy-specific transcriptional changes are not observed during uterine involution, suggesting different underlying molecular mechanisms. These results raise the possibility that activation of atrogin-1 and MuRF-1 may be a common general adaptation in cells undergoing a rapid reduction in size.