Muscle-specific Ubiquitin Research Articles

Unacylated Ghrelin Protects Against Muscle Wasting, Mitochondrial Dysfunction, and Neuromuscular Junction Disruption in Tumor Bearing Mice

Background: Cancer cachexia is a complex metabolic syndrome that severely impacts mobility, treatment strategies, and life quality of the patients. However, no treatments are available to mitigate the debilitating consequences of cancer cachexia. Ghrelin is a hormone released from the stomach that increases appetite upon acylation by binding to GHSR1a receptors in the brain. Because of its orexigenic effects and increases in body weight, ghrelin GHSR1a receptor analogs were tested as clinical trials to mitigate muscle wasting and loss of strength in cancer patients in several countries. The analogs (i.e., Anamorelin) increased body weight of the non-small lung cancer patients in European countries and Japan, but failed to increase strength, which is the critical component in cancer cachexia. Mainly due to the lack of improvement in strength and patients’ quality of life, Anamorelin was not approved for clinical use in Europe. While the lack of increases in strength is, at least in part, associated with the adipogenic effects of acylated ghrelin, unacylated ghrelin directly improves satellite cell renewal, muscle growth, and mitochondrial function independent of GHSR1a receptor activation and increases in appetite. Unacylated ghrelin is the predominant population in circulation comprising proximately 90-95% of total ghrelin in humans and rodents. Hypothesis: Unacylated ghrelin projects against muscle wasting, loss of strength, and metabolic dysfunction of tumor bearing mice. Methods and Results: Supplementing the cancer cell conditioned medium (CM) with unacylated ghrelin preserved myotube size. Unacylated ghrelin also diminished the cancer cell CM-induced upregulation of MuRF-1, muscle specific ubiquitin ligase. Interestingly, unacylated ghrelin normalized transcriptional upregulation of denervation markers (i.e., sarcolipin, Runx1, GADD45a) in myotubes treated with CM. To further investigate the therapeutic effects of unacylated ghrelin in vivo, we inoculated lung cancer cell line (LL2, ATCC®) in the flank of 4 months old male mice. Two weeks after the injection prior to development of cachexia, mice were provided with unacylated ghrelin containing drinking water or untreated water as control. Tumor bearing (TB) mice treated with unacylated ghrelin showed 30-40% increases in gastrocnemius and quadriceps weights, while soleus muscle mass was unchanged. In addition to the muscle quantity, unacylated ghrelin increased force generating capacity was also improved by unacylated ghrelin. Notably, unacylated ghrelin increased mitochondrial oxygen consumption rate and protected against neuromuscular junction disruption and denervation in TB control mice. Conclusion: Unacylated ghrelin protects against cancer cachexia by targeting multiple risk factors in skeletal muscle wasting, including protein balance, metabolic alterations, and neuromuscular junction disruption. Our findings show protective effects of unacylated ghrelin against cachexia and neuromuscular impairment in tumor bearing mice, indicating its therapeutic potential in cancer patients. NIA R00 064143 to Ahn B. Pilot funds from the Center for Redox Biology in Medicine, and Cancer Genetics and Metabolism program at the Wake Forest Baptist Comprehensive Cancer Center. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Therapeutic Consequences of Targeting the IGF-1/PI3K/AKT/FOXO3 Axis in Sarcopenia: A Narrative Review.

The high prevalence of sarcopenia in an aging population has an underestimated impact on quality of life by increasing the risk of falls and subsequent hospitalization. Unfortunately, the application of the major established key therapeutic-physical activity-is challenging in the immobile and injured sarcopenic patient. Consequently, novel therapeutic directions are needed. The transcription factor Forkhead-Box-Protein O3 (FOXO3) may be an option, as it and its targets have been observed to be more highly expressed in sarcopenic muscle. In such catabolic situations, Foxo3 induces the expression of two muscle specific ubiquitin ligases (Atrogin-1 and Murf-1) via the PI3K/AKT pathway. In this review, we particularly evaluate the potential of Foxo3-targeted gene therapy. Foxo3 knockdown has been shown to lead to increased muscle cross sectional area, through both the AKT-dependent and -independent pathways and the reduced impact on the two major downstream targets Atrogin-1 and Murf-1. Moreover, a Foxo3 reduction suppresses apoptosis, activates satellite cells, and initiates their differentiation into muscle cells. While this indicates a critical role in muscle regeneration, this mechanism might exhaust the stem cell pool, limiting its clinical applicability. As systemic Foxo3 knockdown has also been associated with risks of inflammation and cancer progression, a muscle-specific approach would be necessary. In this review, we summarize the current knowledge on Foxo3 and conceptualize a specific and targeted therapy that may circumvent the drawbacks of systemic Foxo3 knockdown. This approach presumably would limit the side effects and enable an activity-independent positive impact on skeletal muscle.

Hypoglycemic drug liraglutide alleviates low muscle mass by inhibiting the expression of MuRF1 and MAFbx in diabetic muscle atrophy.

Low muscle mass, that is, muscular atrophy, is an independent risk factor for type 2 diabetes mellitus (T2DM). Few studies investigated whether hypoglycemic drugs can alleviate low muscle mass and related mechanisms. This study recruited 51 type 2 diabetes mellitus (T2DM) patients, who were divided into two groups based on skeletal muscle index (SMI) evaluated by Dual-energy X-ray absorptiometry (DXA): the experiment group (n = 25, SMI < 7 kg/m 2 ) and the control group (n = 26, SMI≥7 kg/m 2 ). GLP-1 levels were measured by ELISA. In vitro, 10 KK-A y mice (11- to 12-week-old) were assigned into two groups: liraglutide group (n = 5) and saline group (n = 5). Real-time PCR and Western blot were used to determine the expression levels of muscle specific ubiquitin protease E3, MuRF1, and MAFbx. T2DM patients with a higher SMI had significantly higher GLP-1 levels (t = 3.77, p < 0.001). SMI were positively associated with GLP-1 levels (β = 0.435, p = 0.001) and inversely associated with age (β = 0.299, p = 0.015). The incidence of low muscle mass at below the second quartiles was 10.55 times that of above the second quartiles (odds ratio = 10.556, p < 0.001). Liraglutide-treatment mice showed significant decrease in food intake, final body weight, fasting blood glucose, and significant increase in skeletal muscle mass, which coincided with the significant decrease in the expression levels of ubiquitin protease E3 MuRF1 and MAFbx. In vitro studies showed that liraglutide promoted myogenic differentiation and attenuated dexamethasone (DEX)-induced myotube atrophy. Ectopic expression of MuRF1 and MAFbx antagonized the beneficial effects of liraglutide on DEX-induced myotube atrophy. T2DM patients have muscular atrophy, and liraglutide alleviates muscular atrophy at least in part by inhibiting the expression of MuRF1 and MAFbx.

Biophysical and functional study of CRL5Ozz, a muscle specific ubiquitin ligase complex

Ozz, a member of the SOCS-box family of proteins, is the substrate-binding component of CRL5Ozz, a muscle-specific Cullin-RING ubiquitin ligase complex composed of Elongin B/C, Cullin 5 and Rbx1. CRL5Ozz targets for proteasomal degradation selected pools of substrates, including sarcolemma-associated β-catenin, sarcomeric MyHCemb and Alix/PDCD6IP, which all interact with the actin cytoskeleton. Ubiquitination and degradation of these substrates are required for the remodeling of the contractile sarcomeric apparatus. However, how CRL5Ozz assembles into an active E3 complex and interacts with its substrates remain unexplored. Here, we applied a baculovirus-based expression system to produce large quantities of two subcomplexes, Ozz–EloBC and Cul5–Rbx1. We show that these subcomplexes mixed in a 1:1 ratio reconstitutes a five-components CRL5Ozz monomer and dimer, but that the reconstituted complex interacts with its substrates only as monomer. The in vitro assembled CRL5Ozz complex maintains the capacity to polyubiquitinate each of its substrates, indicating that the protein production method used in these studies is well-suited to generate large amounts of a functional CRL5Ozz. Our findings highlight a mode of assembly of the CRL5Ozz that differs in presence or absence of its cognate substrates and grant further structural studies.

Imoxin Prevents Dexamethasone‐Induced Promotion of Muscle‐Specific E3 Ubiquitin Ligases and Stimulates Anabolic Signaling in C2C12 Myotubes

Muscle atrophy is the loss of skeletal muscle mass during several pathological conditions such as long‐term fasting, aging, cancer, diabetes, sepsis and immune disorders. Dexamethasone (DEX), a synthetic glucocorticoid, induces skeletal muscle atrophy by suppression of protein synthesis and promotion of protein degradation. Specifically, DEX increases the levels of muscle specific ubiquitin E3 ligases including MAFbx and MuRF1. The double‐stranded RNA (dsRNA)‐activated protein kinase R (PKR) plays a significant role in mediating inflammation and glucose homeostasis. However, pathological roles of PKR in muscle atrophy are not fully understood. The current study aimed to investigate the effect of imoxin, a PKR inhibitor, on DEX‐induced muscle atrophy in C2C12 myotubes. Myotubes were incubated with imoxin at different concentrations with or without 5 μM DEX for 24 h. Western blotting was performed to measure proteins involved in muscle protein synthesis as well as degradation. DEX significantly augmented the protein levels of MuRF1 and MAFbx (275 ± 5 % and 694 ± 36 %, respectively) compared with serum free control (CON). However, 5 μM imoxin treatment significantly reduced protein levels of MuRF1 by 88 ± 2 % and MAFbx by 99 ± 0 % compared with DEX, respectively. In addition, DEX suppressed FoxO4 phosphorylation (33 ± 9 %) compared to CON, but 5 μM imoxin treatment promoted FoxO4 phosphorylation (735 ± 44 %) compared to DEX. Additionally, DEX stimulated protein ubiquitination compared with control (124 ± 1 %) but 5 μM imoxin treatment reduced protein ubiquitination by 42 ± 4 % compared to DEX. At the same time, 5 μM imoxin treatment stimulated Akt phosphorylation (195 ± 5 %), mTOR phosphorylation (171 ± 21%) and p70S6K1 phosphorylation (314 ± 31 %) under DEX‐treated condition even though DEX treatment did not suppressed Akt/mTOR/p70S6K1 axis. These findings suggest that imoxin protects against DEX‐induced skeletal muscle atrophy by alleviating muscle specific E3 ubiquitin ligases and promoting protein synthesis via Akt/mTOR/S6K1 axis in muscle cells.Support or Funding InformationNone

The muscle-specific MuRF1 ubiquitin ligase transcriptionally regulates cardiomyocyte autophagy in a FOXO1/3-dependent manner and protects against cardiac inflammation in vivo

The muscle-specific MuRF1 ubiquitin ligase transcriptionally regulates cardiomyocyte autophagy in a FOXO1/3-dependent manner and protects against cardiac inflammation in vivo

PO-094 p38 MAPK controls of E3-ligases expression and soleus atrophy attenuation in rat upon hindlimb unloading

Objective Unloading causes rapid skeletal muscle atrophy mainly due to the increased protein degradation. Muscle proteolysis results from the activation of ubiquitin-proteasome systems. The ubiquitination proteins are carried out by muscle-specific E3 ubiquitin ligases – MuRF-1 and MAFbx. It is known that MuRF-1 and MAFbx expression significantly increases on the third day of muscle unloading. We tested the hypothesis that p38 MAPK participates in the regulation of E3 ligases expression and the development of skeletal muscle atrophy during unloading. To check this idea we inhibited p38 MAPK by VX-745.&#x0D; Methods 21 male Wistar rats were divided into 3 groups (7 rats in each group): intact control (C), rats suspended for 3 days (HS) and rats suspended and injected i.p. with VX-745 (10 mg/kg/day) (VX). The hindlimb suspension was carried out according to Morey-Holton technique. The animals were anaesthetised with an i.p. injection of tribromoethanol (240 mg/kg). Under anesthesia, the m.soleus were excised, frozen in liquid nitrogen, and stored at -80°C until further analysis. All procedures with the animals were approved by the Biomedicine Ethics Committee of the Institute of Biomedical Problems of the Russian Academy of Sciences/Physiology section of the Russian Bioethics Committee. The statistical analysis was performed using the REST 2009 v.2.0.12 and Origin Pro programs at the significance level set at 0,05. The results are given as median in percent and interquartile range (0.25-0.75).&#x0D; Results The muscle weight in HS group was significantly reduced (72,3±2,5 mg) compared to C (83,0±3 mg), p&lt;0.05, while the soleus weight of VX group didn’t differ from the control (84.2±5 mg). The MuRF1 mRNA expression was elevated dramatically in HS group (165 (138-210) %) when compared with the control (100 (64.6-112.5) %), p&lt;0.05. In the VX group the level of MuRF1 mRNA expression (127 (105-138) %) didn’t differ from the control group. The MAFbx mRNA expression was observed to increase equally in both suspended groups (294 (265-342) % and (271 (239-309) %).) vs C (100 (91-106) %) so, VX-745 administration did not have any significant effect on its expression. We also found that the level of ubiquitin mRNA expression in the soleus of HS rats was higher (423 (325-485) %) in comparison with the C group (100 (78-166) %, p&lt;0.05) while VX-745 injection prevented increasing the mRNA ubiquitin expression (200 (190-237) %). We discovered that the elevation of calpain-1 mRNA expression upon HS was prevented by VX-745 administration and its level didn’t differ from the control group (C - 100 (97-105) %, HS – 120 (116-133) %, VX - 107 (100-115) %, p&lt;0.05).&#x0D; Conclusions Thus, the results indicate that the p38 MAPK signaling pathway takes part in the regulation of E3-ligase MuRF1 but not MAFbx expression. The p38 MAPK inhibition prevents muscle atrophy and the elevation of ubiquitin and calpain mRNA expression at the early stage of hindlimb unloading. This work was supported by RFBR grant No.17-04-01838.

Sulforaphane prevents dexamethasone-induced muscle atrophy via regulation of the Akt/Foxo1 axis in C2C12 myotubes

Muscle atrophy occurs in various catabolic conditions, including hormone imbalance, severe injury, sepsis, cancer, and aging. Dexamethasone (DEX) is a synthetic glucocorticoid and is used an anti-inflammatory agent. However, when chronically used, it is accompanied by side effects, such as, muscle atrophy, diabetes mellitus, and obesity. In this study, we investigated the effect of sulforaphane (SFN) on DEX-induced muscle atrophy and the underlying mechanisms involved. DEX induced muscle atrophy was accompanied by increased muscle specific ubiquitin E3 ligase markers, such as, Atrogin-1 and myostatin, and decreased MyoD in C2C12 myotubes. To investigate the role played by SFN in DEX-induced muscle atrophy, we quantified mRNA levels of muscle atrophy markers, protein synthesis using a puromycin incorporation assay, protein degradation by ubiquitination, and myotube diameters by PAS staining in C2C12 myotubes co-treated with DEX and SFN. Interestingly, SFN effectively prevented myostatin and Atrogin-1 mRNA upregulations by DEX, increased the mRNA level of MyoD, and consequently, reduced protein degradation. Furthermore, SFN enhanced protein synthesis through a Foxo-dependent pathway by activating Akt, and thus, increased myotube diameters. These results show SFN inhibits DEX-induced muscle atrophy in C2C12 myotubes via Akt/Foxo signaling.

Abstract LB-267: Metabolic alterations in tumors cause cachexia in pancreatic cancer

Abstract Cancer-associated cachexia is a complex metabolic syndrome which leads to excessive loss of skeletal muscle and adipose deposits. Up to 80% of pancreatic cancer patients suffer from cachexia and nearly one third die due to complexities related to the syndrome. Treatment of cachexia will not only improve the standard of living of pancreatic cancer patients but would also improve the patient survival. In the clinic, the majority of cancer patients are diagnosed at the refractory phase of cachexia, which cannot be reversed by dietary interventions. Till late, cachexia has mainly been attributed to systemic inflammation caused by cytokines released from the host and the tumor. But anti-inflammatory therapies have not shown promising results in clinical trials. Hence, there is an urgent need to identify new targets to combat cancer cachexia. It has been previously shown that there are constant metabolic interactions between the host body and the tumor. Increase in tumor burden and its demand for nutrition forces the host body to reprogram its metabolism leading to the breakdown of host body proteins into amino acids, which gets taken up and converted into carbohydrates by the tumor. To study the role of metabolic pathways in tumor cells on cachectic factor secretion, we pre-treated pancreatic cancer cells with metabolic inhibitors 3-Bromopyruvic acid and bis-2-(5-phenylacetomido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide (BPTES). The conditioned media from control and treated cells were then evaluated for myotube degeneration potential in C2C12-dfferentitaed myotubes by measuring myotube thickness, protein content and expression of muscle specific ubiquitin ligases. We observed that that pre-treated conditioned-media had reduced myotubes degenerating potential as compared to controls. Furthermore, we inhibition of glycolytic flux in tumor cells by treatment with ketone bodies also diminished myotubes degenerating potential. Tumor-bearing mice had decreased tumor growth and cachexia when fed on a ketogenic diet. In conclusion, metabolic reprograming of tumor cells plays an important role in inducing pancreatic cancer-induced cachexia and delineating the metabolic pathways is crucial to elucidate important therapeutic targets for cancer cachexia. Citation Format: Aneesha Dasgupta, Surendra K. Shukla, Venugopal Gunda, Nina V. Chaika, Pankaj K. Singh. Metabolic alterations in tumors cause cachexia in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-267. doi:10.1158/1538-7445.AM2017-LB-267

OP-01 - Treatment of sarcopenia by targeting Akt and muscle specific ubiquitin ligases. Evidence from mice and from a clinical trial

Disuse muscle wasting may take place as a result of several such as joint immobilization, inactivity or bed rest. There are no good therapies to treat it. Allopurinol, a drug commonly used to treat hyperuricemia and gout, protects muscle damage, specially after exhaustive exercise and results in functional gains in old persons. Thus, we tested its effect in the prevention of atrophy of the soleus muscle after two weeks of hindlimb unloading in experimental animals (mice), and lower leg immobilization following ankle sprain in humans (Registration of the clinical Trial: EUDRACT2011-003541-17). We have found show that allopurinol protects against muscle atrophy in both mice and humans. The protective effect of allopurinol is similar, or even superior, to that of resistance exercise which is the best-known way to prevent muscle mass loss in disuse human models. We have also found that allopurinol protects against muscle mass loss by inhibiting the expression of ubiquitin ligases. Thus, the ubiquitin-proteasome pathway is an adequate therapeutic target to inhibit muscle wasting and underpins the role of allopurinol as a non-hormonal intervention to treat disuse muscle atrophy.

Acute high-caffeine exposure increases autophagic flux and reduces protein synthesis in C2C12 skeletal myotubes.

Caffeine is a highly catabolic dietary stimulant. High caffeine concentrations (1-10mM) have previously been shown to inhibit protein synthesis and increase protein degradation in various mammalian cell lines. The purpose of this study was to examine the effect of short-term caffeine exposure on cell signaling pathways that regulate protein metabolism in mammalian skeletal muscle cells. Fully differentiated C2C12 skeletal myotubes either received vehicle (DMSO) or 5mM caffeine for 6h. Our analysis revealed that caffeine promoted a 40% increase in autolysosome formation and a 25% increase in autophagic flux. In contrast, caffeine treatment did not significantly increase the expression of the skeletal muscle specific ubiquitin ligases MAFbx and MuRF1 or 20S proteasome activity. Caffeine treatment significantly reduced mTORC1 signaling, total protein synthesis and myotube diameter in a CaMKKβ/AMPK-dependent manner. Further, caffeine promoted a CaMKII-dependent increase in myostatin mRNA expression that did not significantly contribute to the caffeine-dependent reduction in protein synthesis. Our results indicate that short-term caffeine exposure significantly reduced skeletal myotube diameter by increasing autophagic flux and promoting a CaMKKβ/AMPK-dependent reduction in protein synthesis.

Regulation of Cardiac Autophagic Flux In Vivo by the Ubiquitin Ligase Muscle Ring Finger‐1 (MuRF1)

The ubiquitin‐proteasome and autophagy systems are comdplementary protein degradation pathways that play a critical role in protein quality control in cardiomyocytes. Our laboratory studies the muscle specific ubiquitin ligase MuRF1 and have identified that mice with cardiac‐specific increased MuRF1 expression is cardioprotective in cardiac I/R injury, while also more susceptible to heart failure when challenged with pressure overload‐induced cardiac hypertrophy. Since these phenotypes are consistent with increased autophagic flux, we tested the hypothesis that MuRF1 positively regulated cardiac autophagy. We first crossed GFP‐RFP‐LC3 Tg+ mice created to track autophagy with our MuRF1 Tg+ mice to create MuRF1/GFP‐RFP‐LC3 dual Tg+ mice. Using confocal analysis of early (dual green and red) and late (red) punta representing the autophagosome‐autolysosome transition, we identified that MuRF1 Tg+ mice had significantly increased cardiac autophagic flux. MuRF1 Tg+ hearts also had significantly increased LC3II levels by immunoblot post‐bafilomycin treatment indicative of increased autophagic flux. Conversely, MuRF1−/− hearts had significantly decreased LC3II levels by immunoblot analysis, consistent with decreased autophagic flux. We next hypothesized that MuRF1 supported autophagy by post‐translational modification of FoxO1 and FoxO3a, two major transcription factors previously described to regulate autophagy. Immunoblot analysis of the inactive p‐FoxO1 and p‐FoxO3a proteins in MuRF1Tg+ hearts demonstrated decreased p‐FoxO3a (p&lt;0.05), while MuRF1−/− hearts had increased levels (p&lt;0.05), consistent with MuRF1's support of Foxo1/3 activity. To determine if MuRF1 regulated FoxO activity transcriptionally, FoxO3a activity assays were performed by in vitro luciferase assays. Increasing MuRF1 levels were shown to enhance FoxO3a transcriptional activity (p&lt;0.05), offering one potential mechanism by which MuRF1 supports autophagy in vivo. Lastly, we determined if MuRF1‐induced autophagy was cardioprotective in doxorubicin‐induced cardiomyocyte death, a model where increased autophagy has been reported to be protective. When MuRF1 expression was increased in the H9C2 cardiomyocyte‐derived cell line and challenged with 1 microM doxorubicin for 24 hours, a 66% reduction in Caspase 3/7 activity was seen compared to control cells, indicating that MuRF1's cardioprotection may be due, in part, to enhanced autophagy. These findings establish MuRF1 as the first ubiquitin ligase to regulate cardiac autophagy, which occurs via the FoxO3a transcription factor. Understanding MuRF1's regulation of autophagy provides insight for the development of therapies targeting autophagy in heart disease.

Grape seed extract prevents skeletal muscle wasting in interleukin 10 knockout mice.

BackgroundMuscle wasting is frequently a result of cancers, AIDS, chronic diseases and aging, which often links to muscle inflammation. Although grape seed extract (GSE) has been widely used as a human dietary supplement for health promotion and disease prevention primarily due to its anti-oxidative and anti-inflammative effects, it is unknown whether GSE affects muscle wasting. The objective is to test the effects of GSE supplementation on inflammation and muscle wasting in interleukin (IL)-10 knockout mice, a recently developed model for human frailty.MethodsMale IL-10 knockout (IL10KO) C57BL/6 mice at 6 weeks of age were assigned to either 0% or 0.1% GSE (in drinking water) groups (n = 10) for 12 weeks, when skeletal muscle was sampled for analyses. Wild-type C57BL/6 male mice were used as controls.ResultsTibialis anterior muscle weight and fiber size of IL10KO mice were much lower than wild-type mice. IL10KO enhanced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and inflammasome formation when compared to wild-type mice. Phosphorylation of anabolic signaling was inhibited, whereas muscle specific ubiquitin ligase, AMP-activated protein kinase (AMPK) and apoptotic signaling were up-regulated in IL10KO mice. GSE supplementation effectively rectified these adverse changes in IL10KO muscle, which provide an explanation for the enhanced muscle mass, reduced protein degradation and apoptosis in GSE supplemented mice compared to IL10KO mice without supplementation.ConclusionGSE supplementation effectively prevents muscle wasting in IL10KO mice, showing that GSE can be used as an auxiliary treatment for muscle loss associated with chronic inflammation and frailty.

Astaxanthin attenuates lipopolysaccharide‐induced atrogin‐1 expression in C2C12 myotubes (1163.22)

Atrogin‐1, as a muscle specific ubiquitin ligase, is elevated in skeletal muscle during cachectic state, causing muscle atrophy. The elevation of atrogin‐1 expression level is induced by inflammatory cytokines, and they are promoted by oxidative stress. Astaxanthin, a member of carotenoid family, exerts high antioxidative capacity. We investigated preventive effect of astaxanthin on atrogin‐1 expression and muscle atrophy caused by lipopolysaccharide (LPS) treatment. In C2C12 myotubes, LPS treatment markedly increased the level of atrogin‐1 mRNA expression, leading to myotube atrophy. Astaxanthin treatment significantly attenuated the increased atrogin‐1 mRNA expression, and prevented myotube atrophy induced by LPS in C2C12 myotubes. These results suggest the efficacy of astaxanthin could provide a preventive approach against cachexia‐related muscle atrophy.

Abstract 294: Atrogin-1 Is Required for Atrophic Remodeling of the Heart

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1 is a muscle specific ubiquitin ligase required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied. Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program were detected in both WT and Atrogin-1 -/-transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, calcinuerin, a known target of Atrogin-1 involved in hypertrophy and protein synthesis, was upregulated in unloaded Atrogin-1 deficient hearts. Consequently, μunloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. While inhibition of calcineurin decreased rates of protein synthesis in unloaded cardiomyocytes in the absence of Atrogin-1, protein synthesis rates were still higher than in WT unloaded cardiomyocytes. These results suggest that more than one pathway regulating protein synthesis is controlled by Atrogin-1 in the heart. Furthermore, the data provide evidence that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

KBTBD13 interacts with Cullin 3 to form a functional ubiquitin ligase

Autosomal dominant mutations in BTB and Kelch domain containing 13 protein (KBTBD13) are associated with a new type of Nemaline Myopathy (NEM). NEM is a genetically heterogeneous group of muscle disorders. Mutations causing phenotypically distinct NEM variants have previously been identified in components of muscle thin filament. KBTBD13 is a muscle specific protein composed of an N terminal BTB domain and a C terminal Kelch-repeat domain. The function of this newly identified protein in muscle remained unknown. In this study, we show that KBTBD13 interacts with Cullin 3 (Cul3) and the BTB domain mediates this interaction. Using ubiquitination assays, we determined that KBTBD13 participates in the formation of a Cul3 based RING ubiquitin ligase (Cul3-RL) capable of ubiquitin conjugation. Confocal microscopy of transiently expressed KBTBD13 revealed its co-localization with ubiquitin. Taken together, our results demonstrate that KBTBD13 is a putative substrate adaptor for Cul3-RL that functions as a muscle specific ubiquitin ligase, and thereby implicate the ubiquitin proteasome pathway in the pathogenesis of KBTBD13-associated NEM.

Abstract P180: Atrogin-1, a Muscle-Specific Ubiquitin Ligase, Drives Atrophic Remodeling of the Heart

The heart adapts to changes in load by remodeling both metabolically and structurally. During this process, cardiomyocytes break down unnecessary or damaged proteins and use the resulting amino acids for the synthesis of new proteins and/or energy provision. Protein degradation via the ubiquitin proteasome system is controlled by ubiquitin ligases, which determine the specific proteins to be degraded. Atrogin-1, a muscle specific ubiquitin ligase, is required for skeletal muscle atrophy, and over-expressing Atrogin-1 inhibits the development of cardiac hypertrophy. We now tested the hypothesis that Atrogin-1 is required for atrophic remodeling of the unloaded heart. Hearts from wild type (WT) and Atrogin-1 -/- mice (8-10 weeks old, n =8-12) were subjected to mechanical unloading by heterotopic transplantation. In WT hearts, seven days of unloading significantly reduced heart weight and myocyte cross-sectional area, while hearts lacking Atrogin-1 significantly hypertrophied (at least a 1.5-fold increase in heart weight, 2-fold increase in myocyte area). Conventional markers of atrophic remodeling, such as the reactivation of the fetal gene program (ANF, MHC isoform switch), were detected in both WT and Atrogin-1 -/- transplanted hearts. Proteasome activity and markers of autophagy were increased after unloading, although not significantly different between WT and Atrogin-1 -/- hearts. Pathways regulating protein synthesis were enhanced in the absence of Atrogin-1; there was an increase in activated Akt and its downstream pathway including mTOR, 4E-BP1, and p70 S6 kinase. Additionally, two known targets of Atrogin-1 involved in hypertrophy and protein synthesis, calcineurin and eukaryotic initiation factor 3f, were upregulated in unloaded Atrogin-1 deficient hearts. Consequently, “unloaded” cardiomyocytes lacking Atrogin-1 in vitro exhibit increased basal rates of protein synthesis. The results suggest that Atrogin-1 not only enhances protein degradation, but also keeps protein synthesis in check. Thus Atrogin-1 has a duel role in regulating cardiac mass.

Abstract 5276: Exploration of the cachectic phenotype

Abstract The complexities of cancer, and cachexia induced by cancer, dictate the necessity of studying this disease in the context of its microenvironment as well as in the context of interactions between the tumor and the body, i.e., the ‘macroenvironment’. In the present study, we are applying molecular and functional imaging to understand cancer cachexia and develop clinically translatable indices for early detection of this condition. As models, we are using cachectic (MAC16) and non-cachectic (MAC13) murine colon adenocarcinoma cells. MAC16 tumors induce extensive weight loss in tumor-bearing animals, whereas MAC13 tumors, although histologically similar, do not induce weight loss. By using in vivo 1H MRSI, we detected a high level of total choline in cachectic tumors compared to non-cachectic ones but no differences in lactate levels in tumor extracts. We then performed [18F]fluorodeoxyglucose positron emission tomography studies and observed a significant increase in glucose uptake in the cachectic tumors compared to the non-cachectic ones. To detect the early onset of cachexia inducing signals, we have created a cell based optical biosensor using genetically engineered myoblasts that are injected into the gastrocnemius skeletal muscle. Glucocorticoids induce the expression of a muscle specific ubiquitin ligase (MuRF1), which has been shown to be involved with the catabolism of muscle proteins during cachexia. The up-regulation of MuRF1 occurs by the specific activation of the glucocorticoid receptor (GR) upon hormone binding. Activated GR then translocates to the nucleus and directly binds to its response element (GRE) within the MuRF1 promoter. Importantly, increased expression of MuRF1 has been shown to be an early event in cachexia processes. We have constructed a fluorescent protein reporter gene that is linked to a portion of the MuRF1 promoter containing the GRE, to report on the initial stages of the induction of cachexia. This reporter system was tested and optimized in mouse C2C12 myoblast cells, a well-established muscle cell model system. C2C12 myoblast cells were transfected with constitutively expressed green fluorescent protein (GFP) and MURF1 promoter driven tdTomato red fluorescent protein (RFP). RFP expression can then be induced by dexamethasone treatment through MURF1 promoter transactivation by GR. Stably transfected C2C12 myoblasts will be used as cell based optical biosensors. Regulatory changes occurring in engrafted C2C12 muscle cells in cachectic mice will be compared to analogous grafts on non-cachectic mice to identify the sequence of events in the cachexia cascade. These studies are part of our ongoing work to obtain a comprehensive characterization of the ‘cachectic phenotype’ using noninvasive multimodality imaging that will allow us to detect cancer induced cachexia and identify new targets to prevent or reverse this condition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5276. doi:10.1158/1538-7445.AM2011-5276

Attenuation of Muscle Loss and Maintained Growth Response in Aged Mice with a Null Deletion of Muscle Ring Finger 1 (MuRF1)

During aging progressive declines occur in both the force producing capacity and the oxidative capacity of skeletal muscle. Further, aging is associated with a resistance to muscle growth in response to increased loading. Muscle Ring Finger 1 (MuRF1), a muscle specific ubiquitin ligase, is expressed at low levels in resting muscle, but up-regulated under a variety of atrophy-inducing conditions including denervation, disuse, and possibly aging. Deletion of MuRF1 (KO) attenuates the loss of muscle mass during atrophy; however, its role in aging has not been investigated. Thus, the objective of this study was to examine the metabolic, contractile and growth properties of wild type (WT) and MuRF1 KO mice at 6, 18, and 24 months of age. At 24 months WT, but not KO, mice had significantly lower gastrocnemius mass. Morphological analyses revealed significant decreases in fiber cross sectional area and capillary to fiber ratio in 24 month WT, but not KO mice. Muscle growth was examined following 14-days of functional overload of the plantaris in young and old WT and KO mice. Load-induced growth was similar in young WT and KO mice. Old KO mice maintained the growth response observed in young mice, whereas load-induced growth was significantly less in old WT mice. These data suggest that MuRF1 may play an important role in age-related loss of muscle mass and anabolic resistance. Supported by NIH RO1DK75801 and T32HL086350.

Effects of chronic hypoglycemia and euglycemic correction on lysine metabolism in fetal sheep

In this study, we determined rates of lysine metabolism in fetal sheep during chronic hypoglycemia and following euglycemic recovery and compared results with normal, age-matched euglycemic control fetuses to explain the adaptive response of protein metabolism to low glucose concentrations. Restriction of the maternal glucose supply to the fetus lowered the net rates of fetal (umbilical) glucose (42%) and lactate (36%) uptake, causing compensatory alterations in fetal lysine metabolism. The plasma lysine concentration was 1.9-fold greater in hypoglycemic compared with control fetuses, but the rate of fetal (umbilical) lysine uptake was not different. In the hypoglycemic fetuses, the lysine disposal rate also was higher than in control fetuses due to greater rates of lysine flux back into the placenta and into fetal tissue. The rate of CO2 excretion from lysine decarboxylation was 2.4-fold higher in hypoglycemic than control fetuses, indicating greater rates of lysine oxidative metabolism during chronic hypoglycemia. No differences were detected for rates of fetal protein accretion or synthesis between hypoglycemic and control groups, although there was a significant increase in the rate of protein breakdown (P<0.05) in the hypoglycemic fetuses, indicating small changes in each rate. This was supported by elevated muscle specific ubiquitin ligases and greater concentrations of 4E-BP1. Euglycemic recovery after chronic hypoglycemia normalized all fluxes and actually lowered the rate of lysine decarboxylation compared with control fetuses (P<0.05). These results indicate that chronic hypoglycemia increases net protein breakdown and lysine oxidative metabolism, both of which contribute to slower rates of fetal growth over time. Furthermore, euglycemic correction for 5 days returns lysine fluxes to normal and causes an overcorrection of lysine oxidation.