Muscle-specific E3 Ubiquitin Ligases Research Articles

Differential effects of cholecalciferol and calcitriol on muscle proteolysis and oxidative stress in angiotensin II-induced C2C12 myotube atrophy.

Renin-angiotensin system activation contributes to skeletal muscle atrophy in aging individuals with chronic diseases. We aimed to explore the effects of cholecalciferol (VD3) and calcitriol (1,25VD3) on signaling of muscle proteolysis and oxidative stress in myotubes challenged with angiotensin II (AII). The mouse C2C12 myotubes were assigned to vehicle, AII, AII + VD3, AII + 1,25VD3, and AII + losartan groups. The expression levels of muscle-specific E3 ubiquitin ligase proteins, autophagy-related proteins, and oxidative stress markers were investigated. We demonstrated the diverse effects of VD3 and 1,25VD3 on AII-induced myotube atrophy. The myotube diameter was preserved by treatment with 100 nM VD3 and losartan, while 1 and 10 nM 1,25VD3 increased levels of FoxO3a, MuRF1, and atrogin-1 protein expression in myotubes exposed to AII. Treatment with AII + 10 nM 1,25VD3 resulted in the upregulation of LC3B-II, LC3B-II/LC3B-I, and mature cathepsin L, which are autophagic marker proteins. The p62/SQSTM1 protein was downregulated and vitamin D receptor was upregulated after treatment with AII + 10 nM 1,25VD3. A cellular redox imbalance was observed as AII + 10 nM 1,25VD3-induced reactive oxygen species and NADPH oxidase-2 overproduction, and these changes were associated with an inadequate response of antioxidant superoxide dismutase-1 and catalase proteins. Collectively, these findings provide a translational perspective on the role of vitamin D3 in alleviating muscle atrophy related to high levels of AII.

Remedial effects of tilapia skin peptides against dexamethasone-induced muscle atrophy in mice by modulation of AKT/FOXO3a and Sirt1/PGC-1α signaling pathways

Tilapia skin peptides (TSP) possess a range of physiological activities. This study aimed to explore the effects of TSP on Dexamethasone (DEX)-induced muscle atrophy. In vitro, C2C12 myotube myotube diameter and expression levels of the muscle-specific E3 ubiquitin ligases F-box only protein 32 (Atrogin-1) and muscle ring finger protein 1 (MuRF1) challenged with DEX were reversed by TSP. In vivo, DEX was injected subcutaneously to build muscle atrophy model mice. TSP enhanced grip strength, running distance, body lean muscle content, cross-sectional area of the gastrocnemius and tibialis anterior muscles of DEX-induced mice. Moreover, TSP inhibited the expression levels of Atrogin-1 and MuRF1. Mechanically, TSP improved DEX-induced muscle atrophy by regulating the Protein Kinase B α (AKT)/Forkhead box O3 protein (FOXO3a), Sirtuin 1 (Sirt1)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) signaling pathway, and downstream factors such as nuclear respiratory factor (NRF)1/2 and mitochondrial transcription factor A (TFAM).

Beef peptides mitigate skeletal muscle atrophy in C2C12 myotubes through protein degradation, protein synthesis, and the oxidative stress pathway.

This study aimed to investigate the potential of beef peptides (BPs) in mitigating muscle atrophy induced by dexamethasone (DEX) with underlying three mechanisms in vitro (protein degradation, protein synthesis, and the oxidative stress pathway). Finally, the anti-atrophic effect of BPs was enhanced through purification and isolation. BPs were generated using beef loin hydrolyzed with alcalase/ProteAX/trypsin, each at a concentration of 0.67%, followed by ultrafiltration through a 3 kDa cut-off. BPs (10-100 μg mL-1) dose-dependently counteracted the DEX-induced reductions in myotube diameters, differentiation, fusion, and maturation indices (p < 0.05). Additionally, BPs significantly reduced FoxO1 protein dephosphorylation, thereby suppressing muscle-specific E3 ubiquitin ligases such as muscle RING-finger containing protein-1 and muscle atrophy F-box protein in C2C12 myotubes at concentrations exceeding 25 μg mL-1 (p < 0.05). BPs also enhanced the phosphorylation of protein synthesis markers, including mTOR, 4E-BP1, and p70S6K1, in a dose-dependent manner (p < 0.05) and increased the mRNA expression of antioxidant enzymes. Fractionated peptides derived from BPs, through size exclusion and polarity-based fractionation, also demonstrated enhanced anti-atrophic effects compared to BPs. These peptides downregulated the mRNA expression of primary muscle atrophy markers while upregulated that of antioxidant enzymes. Specifically, peptides GAGAAGAPAGGA (MW 924.5) and AFRSSTKK (MW 826.4) were identified from fractionated peptides of BPs. These findings suggest that BPs, specifically the peptide fractions GAGAAGAPAGGA and AFRSSTKK, could be a potential strategy to mitigate glucocorticoid-induced skeletal muscle atrophy by reducing the E3 ubiquitin ligase activity.

The Anti-Muscle Atrophy Effects of Ishige sinicola in LPS-Induced C2C12 Myotubes through Its Antioxidant and Anti-Inflammatory Actions

Inflammation and oxidative stress are known to be major factors in muscle atrophy. The objective of this study was to evaluate whether the antioxidant activity of Ishige sinicola ethanol extract (ISE) and fractions from ISE could prevent lipopolysaccharide (LPS)-induced muscle atrophy in C2C12 myotubes. IS was extracted with ethanol and fractionated with five organic solvents. Then, ISE and five fractions from ISE were used to evaluate the total antioxidant activity and the protective effect of LPS-induced muscle atrophy in C2C12 myotubes. The ISE and butanol (BuOH) fraction showed higher total antioxidant activity and higher total phenol content than other fractions of ISE. The ISE and BuOH fraction significantly attenuated the LPS-induced diameter of C2C12 myotubes as well as the mRNA and protein expression levels of the muscle-specific E3 ubiquitin ligases. The mRNA expression of forkhead box O type 3α was stimulated by LPS, which was suppressed by the BuOH fraction but not ISE. Furthermore, ISE and the BuOH fraction significantly reduced LPS-stimulated gene expression of pro-inflammatory cytokines and inflammation-inducible enzymes, which was mediated by through the inhibition of the p38/extracellular signal-regulated kinase signaling pathway. Thus, ISE exerts a protective effect against muscle atrophy in LPS-induced C2C12 myotubes through the antioxidant activity and anti-inflammatory effects of ISE.

TESTOSTERONE INDUCES POSTMENOPAUSAL HYPERTENSIVE HYPERTROPHY THROUGH INHIBITING THE ACTIVATION OF AMPK- FOXO1/MURF1 SIGNALING PATHWAY

Objective: It has been found that relative elevated level of testosterone could exacerbates postmenopausal hypertensive left ventricular hypertrophy(PM-HTN-LVH), but the mechanisms responsible are unknown. Moreover, MuRF1, a muscle-specific E3 ubiquitin ligase in the ubiquitination process, was newly identified as a negative regulatory factor of myocardial and muscle tissue in exerting a protective functions on the cardiomyocyte hypertrophy. There are little study to certify whether MuRF1 took part in testosterone-induced PM-HTN-LVH. Design and method: Femal spontaneously hypertensive rats (SHR) were ovariectomized (OVX group) or sham-operated (Sham group) at 12 weeks and then the OVX group were intramuscularly injected the testosterone (T 2.85 mg/kg/ quaque omni die im) (OVX+T group) or vehicle (OVX+ vehicle group) for 4 weeks. Futher, Cultured cardiomyocytes were respectively stimulated with different concentration gradients of testosterone for 24h, then measured the expression of myosin heavy chain (MHC) and MuRF1, as well as the phosphorylation levels of AMPK(p-AMPK) and Forkhead box O 1 (p-FOXO1), were separately measured using western blot. Results: After 4 weeks of testosterone supplementation, SHR heart exhibited significant increments in the heart weight/tibial length and the levels of IVST, LVPWT, LVM and the expression of MHC, and have a lower expression of the MuRF1, p-AMPK and p-FOXO1 in the OVX+T group, compared with OVX group and Sham group. Moreover, the expression level of p-AMPK and p-FOXO1 in the heart were at least twice as many as gastrocnemius muscle among the three group, but the MuRF1 was equivalent between heart and gastrocnemius muscle. In vivo, after the treatment of different gradients of testosterone for 24h, the protein expression of MHC showed an S-shaped growth trend and MuRF1, p-AMPK and p-FOXO1 presented with reverse S-shaped reduction along with increasing of testosterone concentration, peaking at 10-6M testosterone. Conclusions: The present study demonstrates that testosterone induces PM-HTN-LVH by modulating the atrophy related AMPK-FOXO1/MuRF1 signaling pathway in vitro and vivo. Increased the heart expression of MuRF1 by activation of AMPK may reverse testosterone-mediated PM-HTN-LVH, which might cause little change of MuRF1 expression in the skeletal muscle resulting in the muscle atrophy.

Blocking muscle wasting via deletion of the muscle-specific E3 ligase MuRF1 impedes pancreatic tumor growth

Cancer-induced muscle wasting reduces quality of life, complicates or precludes cancer treatments, and predicts early mortality. Herein, we investigate the requirement of the muscle-specific E3 ubiquitin ligase, MuRF1, for muscle wasting induced by pancreatic cancer. Murine pancreatic cancer (KPC) cells, or saline, were injected into the pancreas of WT and MuRF1-/- mice, and tissues analyzed throughout tumor progression. KPC tumors induces progressive wasting of skeletal muscle and systemic metabolic reprogramming in WT mice, but not MuRF1-/- mice. KPC tumors from MuRF1-/- mice also grow slower, and show an accumulation of metabolites normally depleted by rapidly growing tumors. Mechanistically, MuRF1 is necessary for the KPC-induced increases in cytoskeletal and muscle contractile protein ubiquitination, and the depression of proteins that support protein synthesis. Together, these data demonstrate that MuRF1 is required for KPC-induced skeletal muscle wasting, whose deletion reprograms the systemic and tumor metabolome and delays tumor growth.

Abstract 361: PDK4 is dispensable for PDAC chachexia

Abstract Cachexia is an involuntary weight loss and muscle wasting that affects up to 80% of advanced cancer patients. It is an independent prognostic marker for response to treatment and overall survival and accounts for 20% of cancer-associated deaths. Skeletal muscles of cachectic patients manifest metabolic abnormalities, notably increased dependence on lipids as a source of energy at the expense of glucose. Pyruvate Dehydrogenase Kinase 4 (PDK4) is upregulated in the muscles of patients and mice with cancer cachexia. PDK4 inhibits the enzyme responsible for the conversion of pyruvate to acetyl coenzyme A which is further oxidized in the tricarboxylic acid cycle. As a result, lipids become the major substrate for oxidative metabolism at the expense of glucose. Recent reports suggested that PDK4 is responsible for metabolic dysregulation in cachexia, proposing PDK4 as a potential drug target. Here, we hypothesized that the deletion of PDK4 would protect against muscle wasting in an orthotopic model of pancreatic ductal adenocarcinoma (PDAC) by countering metabolic abnormalities driven by PDK4. Methods: 50,000 KPC (LSL-KrasG12D:LSL-Trp53R172H:Pdx1-Cre) cells were injected into the pancreas of male PDK4 knockout mice or wild-type controls. For each genotype, a sham surgery group without implantation was included. Body weight was measured at baseline and later every other day starting from day 7 after implantation. Body composition, i.e., lean and fat mass, was assessed at baseline and then once a week using EchoMRI. In-vivo muscle force production measured by the Aurora Scientific system and locomotor activity measured by Auto-Track were used as readouts of muscle function. Tissue was harvested at early and late stages of disease progression representing early and late cachexia. All measurements were normalized to initial body weight. Two-way ANOVA or Student’s t-test was used for statistical analysis as appropriate. Results: Consistent with previous results, PDAC cachexia induced an increase in the expression of PDK4 in muscle both at mRNA and protein. Expression of other members of the PDK family did not change with cachexia or genotype. Mice with KPC tumors produced less muscle force (-7%, p = 0.03), walked less distance (-30%), and had a longer resting time (17%), p &amp;lt; 0.05 without difference between genotypes. At euthanasia, both genotypes had similar tumor burdens (p &amp;gt;0.9) and exhibited similar degrees of muscle (-13 and -17%, p &amp;lt; 0.0001) and adipose tissue wasting (-50%, p &amp;lt; 0.0001). Deletion of PDK4 did not protect against induction of the muscle-specific E3 ubiquitin ligases in PDAC: FBXO32 and TRIM63 (10.7 fold and 13.6 fold on average, respectively, p &amp;lt; 0.01), molecular hallmarks of muscle atrophy. Overall, our work shows that the knockout of PDK4 does not protect against the development of PDAC cachexia suggesting that PDK4 might be a marker of cachexia but not necessarily a driving mechanism. Citation Format: Omnia U. Gaafer, Joseph Rupert, Joshua Huot, Daenique Jengelley, Andrea Bonetto, Leonidas Koniaris, Teresa Zimmers. PDK4 is dispensable for PDAC chachexia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 361.

Urotensin II can Induce Skeletal Muscle Atrophy Associated with Upregulating Ubiquitin-Proteasome System and Inhibiting the Differentiation of Satellite Cells in CRF Mice.

Skeletal muscle wasting and atrophy is highly prevalent in chronic renal failure (CRF) and increases the risk of mortality. According to our previous study, we speculate that urotensin II (UII) can induce skeletal muscle atrophy by upregulating ubiquitin-proteasome system(UPS) in CRF. C2C12 mouse myoblast cells were differentiated into myotubes, and myotubes were exposed to different concentrations of UII. Myotube diameters, myosin heavy chain(MHC), p-Fxo03A, skeletal muscle-specific E3 ubiquitin ligases such as muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx/atrogin1) were detected. Three animal models (the sham operation mice as normal control (NC) group, wild-type C57BL/6 mice with 5/6 nephrectomy (WT CRF) group, UII receptor gene knock out (UT KO) mice with 5/6 nephrectomy (UT KO CRF) group) were designed. Cross-sectional area (CSA) of skeletal muscle tissues in three animal models were measured, and western blot detected protein of UII, p-Fxo03A, MAFbx and MuRF1, and immunofluorescence assays explored the satellite cell marker of Myod1 and Pax7, and PCR arrays detected the muscle protein degradation genes, protein synthesis genes and the genes which were involved in muscle components. UII could decrease mouse myotube diameters, and upregulate dephosphorylated Fxo03A protein. MAFbx and MuRF1 were higher in WT CRF group than that in NC group, but after UII receptor gene was knocked out (UT KO CRF), their expressions were downregulated. UII could inhibit the expression of Myod1 but not Pax7 in animal study. We first demonstrate that skeletal muscle atrophy induced by UII associated with upregulating ubiquitin-proteasome system and inhibiting the differentiation of satellite cells in CRF mice.

Preventing muscle wasting: pro-insulin C-peptide prevents loss in muscle mass in streptozotocin-diabetic rats.

C-peptide therapy exerts several positive actions on nerves, vasculature, smooth muscle relaxation, kidney function and bone. To date, the role of C-peptide in preventing type 1 diabetes-related muscle atrophy has not been investigated. Our aim was to evaluate if C-peptide infusion prevents muscle wasting in diabetic rats. Twenty-three male Wistar rats were randomly divided into three groups: normal control group, diabetic group and diabetic group plus C-peptide. Diabetes was induced by streptozotocin injection, and C-peptide was administered subcutaneously for 6weeks. The blood samples were obtained at baseline, before streptozotocin injection and at the end of the study to assess C-peptide, ubiquitin and other laboratory parameters. We also tested the ability of C-peptide to regulate the skeletal muscle mass, the ubiquitin-proteasome system, the autophagy pathway as well as to improve muscle quality. C-peptide administration reversed hyperglycaemia (P=0.02) and hypertriglyceridaemia (P=0.01) in diabetic plus C-peptide rats compared with diabetic control rats. The diabetic-control animals displayed a lower weight of the muscles in the lower limb considered individually than the control rats and the diabetic plus C-peptide rats (P=0.03; P=0.03; P=0.04; P=0.004, respectively). The diabetic-control rats presented a significantly higher serum concentration of ubiquitin compared with the diabetic plus C-peptide and the control animals (P=0.02 and P=0.01). In muscles of the lower limb, the pAmpk expression was higher in the diabetic plus C-peptide than the diabetic-control rats (in the gastrocnemius, P=0.002; in the tibialis anterior P=0.005). The protein expression of Atrogin-1 in gastrocnemius and tibialis was lower in the diabetic plus C-peptide than in diabetic-control rats (P=0.02, P=0.03). After 42days, the cross-sectional area in the gastrocnemius of the diabetic plus C-peptide group had been reduced by 6.6% while the diabetic-control rats had a 39.5% reduction compared with the control animals (P=0.02). The cross-sectional area of the tibialis and the extensor digitorum longus muscles was reduced, in the diabetic plus C-peptide rats, by 10% and 11%, respectively, while the diabetic-control group had a reduction of 65% and 45% compared with the control animals (both P<0.0001). Similar results were obtained for the minimum Feret's diameter and perimeter. C-peptide administration in rats could protect skeletal muscle mass from atrophy induced by type 1 diabetes mellitus. Our findings could suggest that targeting the ubiquitin-proteasome system, Ampk and muscle-specific E3 ubiquitin ligases such as Atrogin-1 and Traf6 may be an effective strategy for molecular and clinical intervention in the muscle wasting pathological process in T1DM.

Restricted feeding regimens improve white striping associated muscular defects in broiler chickens.

The current study investigated the effects of intermittent feeding (IF) and fasting strategies at different times post-hatch on muscle growth and white striping (WS) breast development. In the first trial, 32 one-day-old Abor Acre broilers were fed ad libitum (AL) for 3d post-hatch and then randomly allotted into 4 feeding strategies including AL, 1h-IF group (1h IF, 4 times feeding/d, 1h each time), 1.5h-IF (1.5h IF, 4 times feeding/d, 1.5h each time), and fasting (1d acute fasting, 6d free access to feed) groups and fed for 7d. Although angiogenic genes including VEGFA, VEGFR1, and VEGFR2, and myogenic genes including MYOG and MYOD were upregulated (P<0.05), the breast muscle satellite cell (SC) number and PAX7, MYF5 expression were decreased by the IF strategies (P<0.05). One-day fasting at 6d of age also upregulated angiogenic genes and MYOD expression (P<0.05), downregulated MYF5 expression (P<0.05), but did not change SC number (P>0.05). In the second trial, 384 one-day-old birds were fed AL for 1wk and then randomly allotted to the above 4 feeding strategies starting at 8d of age until 42d of age. Similarly, IF and fasting strategies upregulated the expression of angiogenic and myogenic genes (P<0.05). Both 1h-IF and 1.5h-IF increased breast muscle SC number (P<0.05). At slaughter, breast muscle fiber diameter of 1.5h-IF was smaller but the SC number was larger than that of the birds fed AL (P<0.05). The IF and fasting strategies prevented WS development, and reduced breast WS scores and triglyceride content (P<0.05) without changing the body weight (P>0.05). Fasting and 1h-IF reduced the expression of adipogenic genes ZNF423 and PDGFRα (P<0.05). Moreover, IF and fasting strategies reduced fibrosis in breast muscle and reduced skeletal muscle-specific E3 ubiquitin ligases (TRIM63 and MAFBX) (P<0.05). Fasting significantly reduced CASPASE-3 in breast muscle (P<0.05). In conclusion, IF starting in the first week decreases SC number. Compared to AL, IF or fasting promotes muscular angiogenesis, increases SC number, prevents muscle degeneration, and prevents the development of WS without impairing the growth performance of broiler chickens.

Protein therapy of skeletal muscle atrophy and mechanism by angiogenic factor AGGF1.

Skeletal muscle atrophy is a common condition without a pharmacologic therapy. AGGF1 encodes an angiogenic factor that regulates cell differentiation, proliferation, migration, apoptosis, autophagy and endoplasmic reticulum stress, promotes vasculogenesis and angiogenesis and successfully treats cardiovascular diseases. Here, we report the important role of AGGF1 in the pathogenesis of skeletal muscle atrophy and attenuation of muscle atrophy by AGGF1. In vivo studies were carried out in impaired leg muscles from patients with lumbar disc herniation, two mouse models for skeletal muscle atrophy (denervation and cancer cachexia) and heterozygous Aggf1+/- mice. Mouse muscle atrophy phenotypes were characterized by body weight and myotube cross-sectional areas (CSA) using H&E staining and immunostaining for dystrophin. Molecular mechanistic studies include co-immunoprecipitation (Co-IP), western blotting, quantitative real-time PCR analysis and immunostaining analysis. Heterozygous Aggf1+/- mice showed exacerbated phenotypes of reduced muscle mass, myotube CSA, MyHC (myosin heavy chain) and α-actin, increased inflammation (macrophage infiltration), apoptosis and fibrosis after denervation and cachexia. Intramuscular and intraperitoneal injection of recombinant AGGF1 protein attenuates atrophy phenotypes in mice with denervation (gastrocnemius weight 81.3±5.7mg vs. 67.3±5.1mg for AGGF1 vs. buffer; P<0.05) and cachexia (133.7±4.7 vs. 124.3±3.2; P<0.05). AGGF1 expression undergoes remodelling and is up-regulated in gastrocnemius and soleus muscles from atrophy mice and impaired leg muscles from patients with lumbar disc herniation by 50-60% (P<0.01). Mechanistically, AGGF1 interacts with TWEAK (tumour necrosis factor-like weak inducer of apoptosis), which reduces interaction between TWEAK and its receptor Fn14 (fibroblast growth factor-inducing protein 14). This leads to inhibition of Fn14-induced NF-kappa B (NF-κB) p65 phosphorylation, which reduces expression of muscle-specific E3 ubiquitin ligase MuRF1 (muscle RING finger 1), resulting in increased MyHC and α-actin and partial reversal of atrophy phenotypes. Autophagy is reduced in Aggf1+/- mice due to inhibition of JNK (c-Jun N-terminal kinase) activation in denervated and cachectic muscles, and AGGF1 treatment enhances autophagy in two atrophy models by activating JNK. In impaired leg muscles of patients with lumbar disc herniation, MuRF1 is up-regulated and MyHC and α-actin are down-regulated; these effects are reversed by AGGF1 by 50% (P<0.01). These results indicate that AGGF1 is a novel regulator for the pathogenesis of skeletal muscle atrophy and attenuates skeletal muscle atrophy by promoting autophagy and inhibiting MuRF1 expression through a molecular signalling pathway of AGGF1-TWEAK/Fn14-NF-κB. More importantly, the results indicate that AGGF1 protein therapy may be a novel approach to treat patients with skeletal muscle atrophy.

ODP201 Fyn regulates sarcopenia via autophagy

Abstract Sarcopenia is an important element that impairs daily activity. Sarcopenia is also linked to insulin resistance, and the number of diabetic patients also presenting sarcopenia is increasing. In recent years, autophagy has been identified as one of the novel mechanisms associated with sarcopenia. We previously reported that Fyn not only participates in the metabolic syndrome but also that autophagy-regulating sarcopenia was decreased in muscle specific Fyn transgenic mice through STAT3 regulation (Cell metabolism 2010, Cell Rep. 2012). More recently, we revealed Fyn-dependent STAT3 phosphorylation by IL6, which is involved in chronic inflammation and metabolic syndrome, using mouse C2C12 myotube cells expressing shRNA for Fyn. Furthermore, using WT mice with both the denervated and the hind limb suspension (HS) that promotes disuse atrophy by suspending the hind limb, not only both Fyn and IL6 gene expressions were increased but the expression and phosphorylation levels of STAT3 were also augmented, while the autophagic activity was decreased. To confirm those regulations are Fyn dependent, we took advantage of Fyn null mice (FynKO) with HS. Compare to WT mice with HS, all the muscles of FynKO were resistant for the loss of muscle mass induced by HS. The expressions of bothatrogin-1 and muRF-1,the key muscle specific E3 ubiquitin ligases as well as the markers for sarcopenia, were not changed in the muscles of FynKO with HS. Moreover, the STAT3 phosphorylation on tyrosine 705 was decreased in the muscle of FynKO compared to WT mice after hind limb suspension, while expression levels of STAT3 were not changed. IL-6mRNA expression levels were increased in the HS muscle of control mice, but were not changed in FynKO HS muscle. Finally, the autophagic activity, examined by both LC3-2 protein levels after intraperitoneal administration of colchicine andSQSTM1/p62 immunofluorescence in muscles, was retained in FynKO after HS. These results strongly suggest that Fyn is involved not only in the metabolic syndrome but also in the pathogenesis of sarcopenia, and may lead to a better understanding of the pathology of sarcopenia-associated obesity and the development of therapeutic methods. Presentation: No date and time listed

Molecular and cellular effects of gold nanoparticles treatment in experimental diabetic myopathy

BackgroundThis study aims to address the effects of gold nanoparticles (AuNPs) on diabetic myopathy in streptozotocin (STZ)-induced diabetic rats. Materials and methodsAdult male rats were separated into three groups (n = 15): non-diabetic control (ND), diabetic (D), and diabetic treated with AuNPs (2.5 mg/kg, D + AuNPs) intraperitoneally for 4 weeks. A single injection of 50 mg/kg STZ was used to induce diabetes. ResultsTreatment with AuNPs lowered blood glucose levels. Skeletal muscle mRNA expression of two muscle-specific E3 ubiquitin-ligases enzymes, F-box-only protein 32 (FBXO32) and muscle RING-finger protein-1 (MuRF1) were upregulated in the D group. Diabetic rats showed significant increases in the skeletal muscle expression levels of plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), and a decrease in glucose transporter 4 (GLUT4) expression. Superoxide dismutase (SOD) activity decreased and malondialdehyde (MDA) level increased in skeletal muscles of D group. Compared to the D group, expression levels of FBXO32, MuRF1, PAI-1 TNF-α, and TGF-β1 were decreased in the D + AuNPs group, and mRNA of GLUT4 increased. Furthermore, in D + AuNPs group, skeletal muscle MDA levels decreased while SOD activity increased. ConclusionIn experimental models, AuNPs can ameliorate muscle atrophy by reducing hyperglycemia, inflammation, and oxidative stress, and by suppressing the ubiquitin-proteasome proteolytic process.

H2S Protects Against Immobilization-Induced Muscle Atrophy via Reducing Oxidative Stress and Inflammation.

Chronic inflammation and oxidative stress are major triggers of the imbalance between protein synthesis and degradation during the pathogenesis of immobilization-induced muscle atrophy. This study aimed to elucidate the effects of hydrogen sulfide (H2S), a gas transmitter with potent anti-inflammatory and antioxidant properties, on immobilization-induced muscle atrophy. Mice were allocated to control and immobilization (IM) groups, which were treated with slow (GYY4137) or rapid (NaHS) H2S releasing donors for 14 days. The results showed that both GYY4137 and NaHS treatment reduced the IM-induced muscle loss, and increased muscle mass. The IM-induced expressions of Muscle RING finger 1 (MuRF1) and atrogin-1, two muscle-specific E3 ubiquitin ligases, were decreased by administration of GYY4137 or NaHS. Both GYY4137 and NaHS treatments alleviated the IM-induced muscle fibrosis, as evidenced by decreases in collagen deposition and levels of tissue fibrosis biomarkers. Moreover, administration of GYY4137 or NaHS alleviated the IM-induced infiltration of CD45 + leukocytes, meanwhile inhibited the expressions of the pro-inflammatory biomarkers in skeletal muscles. It was found that administration of either GYY4137 or NaHS significantly attenuated immobilization-induced oxidative stress as indicated by decreased H2O2 levels and 8-hydroxy-2′-deoxyguanosine (8-OHdG) immunoreactivity, as well as increased total antioxidant capacity (T-AOC), nuclear factor erythroid-2-related factor 2 (NRF2) and NRF2 downstream anti-oxidant targets levels in skeletal muscles. Collectively, the present study demonstrated that treatment with either slow or rapid H2S releasing donors protected mice against immobilization-induced muscle fibrosis and atrophy. The beneficial effects of H2S on immobilization-induced skeletal muscle atrophy might be due to both the anti-inflammatory and anti-oxidant properties of H2S.

Supplementing cultured human myotubes with hibernating bear serum results in increased protein content by modulating Akt/FOXO3a signaling.

Hibernating bears remain in their dens for 5–7 months during winter and survive without eating or drinking while staying inactive. However, they maintain their physical functions with minimal skeletal muscle atrophy and metabolic dysfunction. In bears, resistance to skeletal muscle atrophy during hibernation is likely mediated by seasonally altered systemic factors that are independent of neuromuscular activity. To determine whether there are components in bear serum that regulate protein and energy metabolism, differentiated human skeletal muscle cells were treated with bear serum (5% in DMEM/Ham’s F-12, 24 h) collected during active summer (July) and hibernating winter (February) periods. The serum samples were collected from the same individual bears (Ursus thibetanus japonicus, n = 7 in each season). Total protein content in cultured skeletal muscle cells was significantly increased following a 24 h treatment with hibernating bear serum. Although the protein synthesis rate was not altered, the expression of MuRF1 protein, a muscle-specific E3 ubiquitin ligase was significantly decreased along with a concomitant activation of Akt/FOXO3a signaling. Increased levels of insulin-like growth factor-1 (IGF-1) were also observed in hibernating bear serum. These observations suggest that protein metabolism in cultured human myotubes may be altered when incubated with hibernating bear serum, with a significant increase in serum IGF-1 and diminished MuRF1 expression, a potential target of Akt/FOXO3a signaling. A protein sparing phenotype in cultured muscle cells by treatment with hibernating bear serum holds potential for the development of methods to prevent human muscle atrophy and related disorders.

Differential Regulation of Myocardial E3 Ligases and Deubiquitinases in Ischemic Heart Failure.

The pathological changes of ubiquitination and deubiquitination following myocardial infarction (MI) and chronic heart failure (CHF) have been sparsely examined. We investigated the expression of muscle-specific E3 ubiquitin ligases and deubiquitinases in MI and CHF. Therefore, mice were assigned to coronary artery ligation for 3 days or 10 weeks as well as for sham operation (each n = 10). Expression of E3 ligases (MAFBX, MURF1, CHIP, ITCH, MDM2) and deubiquitinases (A20, CYLD, UCH-L1, USP14, USP19) was determined. After MI and in CHF, the mRNA expression of MURF1, CHIP and MDM2 (all p < 0.05) was decreased. Protein expression analyses revealed that ITCH expression decreased in CHF (p = 0.01), whereas MDM2 expression increased in MI (p = 0.02) and decreased in CHF (p = 0.02). Except for USP19 mRNA expression that decreased at 3 days and 10 weeks (both p < 0.01), the expression of other deubiquitinases remained unaffected after MI and CHF. The expression of myocardial E3 ligases is differentially regulated following MI, raising the question of whether an upstream regulation exists that is activated by MI for tissue protection or whether the downregulation of E3 ligases enables myocardial hypertrophy following MI.

Systemic ablation of vitamin D receptor leads to skeletal muscle glycogen storage disorder in mice.

BackgroundVitamin D deficiency leads to pathologies of multiple organ systems including skeletal muscle. Patients with severe vitamin D deficiency exhibit muscle weakness and are susceptible to frequent falls. Mice lacking a functional vitamin D receptor (VDR) develop severe skeletal muscle atrophy immediately after weaning. But the root cause of myopathies when vitamin D signalling is impaired is unknown. Because vitamin D deficiency leads to metabolic changes as well, we hypothesized that the skeletal muscle atrophy in mice lacking VDR may have a metabolic origin.MethodsWe analysed wild‐type (WT) mice as well as vitamin D receptor null (vdr−/−) mice for skeletal muscle proteostasis, energy metabolism, systemic glucose homeostasis, and muscle glycogen levels. Dysregulation of signalling pathways as well as the glycogen synthesis and utilization machinery were also analysed using western blots. qRT–PCR assays were performed to understand changes in mRNA levels.ResultsSkeletal muscles of vdr−/− exhibited higher expression levels of muscle‐specific E3 ubiquitin ligases and showed increased protein ubiquitination, suggesting up‐regulation of protein degradation. Foxo1 transcription factor was activated in vdr−/− while Foxo3 factor was unaffected. Fasting protein synthesis as well as mTORC1 pathways were severely down‐regulated in vdr−/− mice. Skeletal muscle ATP levels were low in vdr−/− (0.58 ± 0.18 μmol/mL vs. 1.6 ± 0.0.14 μmol/mL, P = 0.006), leading to increased AMPK activity. Muscle energy deprivation was not caused by decreased mitochondrial activity as we found the respiratory complex II activity in vdr−/− muscles to be higher compared with WT (0.29 ± 0.007 mU/μL vs. 0.16 ± 0.005 mU/μL). vdr−/− mice had lower fasting blood glucose levels (95 ± 14.5 mg/dL vs. 148.6 ± 6.1 mg/dL, P = 0.0017) while they exhibited hyperlactataemia (7.42 ± 0.31 nmol/μL vs. 4.95 ± 0.44 nmol/μL, P = 0.0032), suggesting systemic energy deficiency in these mice. Insulin levels in these mice were significantly lower in response to intraperitoneal glucose injection (0.69 ± 0.08 pg/mL vs. 1.11 ± 0.09 pg/mL, P = 0.024). Skeletal muscles of these mice exhibit glycogen storage disorder characterized by increased glycogen accumulation. The glycogen storage disorder in vdr−/− muscles is driven by increased glycogen synthase activity and decreased glycogen phosphorylase activity. Increased glycogenin expression supports higher levels of glycogen synthesis in these muscles.ConclusionsThe results presented show that lack of vitamin D signalling leads to a glycogen storage defect in the skeletal muscles, which leads to muscle energy deprivation. The inability of vdr−/− skeletal muscles to use glycogen leads to systemic defects in glucose homeostasis, which in turn leads to proteostasis defects in skeletal muscles and atrophy.

HDAC4 Knockdown Alleviates Denervation-Induced Muscle Atrophy by Inhibiting Myogenin-Dependent Atrogene Activation.

Denervation can activate the catabolic pathway in skeletal muscle and lead to progressive skeletal muscle atrophy. At present, there is no effective treatment for muscle atrophy. Histone deacetylase 4 (HDAC4) has recently been found to be closely related to muscle atrophy, but the underlying mechanism of HDAC4 in denervation-induced muscle atrophy have not been described clearly yet. In this study, we found that the expression of HDAC4 increased significantly in denervated skeletal muscle. HDAC4 inhibition can effectively diminish denervation-induced muscle atrophy, reduce the expression of muscle specific E3 ubiquitin ligase (MuRF1 and MAFbx) and autophagy related proteins (Atg7, LC3B, PINK1 and BNIP3), inhibit the transformation of type I fibers to type II fibers, and enhance the expression of SIRT1 and PGC-1 α. Transcriptome sequencing and bioinformatics analysis was performed and suggested that HDAC4 may be involved in denervation-induced muscle atrophy by regulating the response to denervation involved in the regulation of muscle adaptation, cell division, cell cycle, apoptotic process, skeletal muscle atrophy, and cell differentiation. STRING analysis showed that HDAC4 may be involved in the process of muscle atrophy by directly regulating myogenin (MYOG), cell cycle inhibitor p21 (CDKN1A) and salt induced kinase 1 (SIK1). MYOG was significantly increased in denervated skeletal muscle, and MYOG inhibition could significantly alleviate denervation-induced muscle atrophy, accompanied by the decreased MuRF1 and MAFbx. MYOG overexpression could reduce the protective effect of HDAC4 inhibition on denervation-induced muscle atrophy, as evidenced by the decreased muscle mass and cross-sectional area of muscle fibers, and the increased mitophagy. Taken together, HDAC4 inhibition can alleviate denervation-induced muscle atrophy by reducing MYOG expression, and HDAC4 is also directly related to CDKN1A and SIK1 in skeletal muscle, which suggests that HDAC4 inhibitors may be a potential drug for the treatment of neurogenic muscle atrophy. These results not only enrich the molecular regulation mechanism of denervation-induced muscle atrophy, but also provide the experimental basis for HDAC4-MYOG axis as a new target for the prevention and treatment of muscular atrophy.

Identification of the MuRF1 Skeletal Muscle Ubiquitylome Through Quantitative Proteomics.

MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine whether MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.

Identifying a New Mechanism of Sarcopenia by Autophagy

Sarcopenia is one of the critical factors in reducing Activity of Daily Life and associated with morbidity and mortality. Sarcopenia has also been linked to metabolic syndrome. In recent years, it has been reported that autophagy is one of the mechanisms as a cause of sarcopenia. Therefore, we focused on autophagy as a system that can regulate both sarcopenia and metabolic syndrome in skeletal muscle and revealed that non-receptor tyrosine kinase Fyn not only participates in metabolic syndrome but also regulates autophagy regulating sarcopenia through STAT3 regulation, mainly using transgenic mice (Cell metabolism 2010, Cell Rep. 2012). However, since these were non-physiological studies, we proceeded with further studies and demonstrating that Fyn dependent STAT3 phosphorylation by IL6, which is involved in chronic inflammation and metabolic syndrome, was observed in mouse C2C12 myotube cells. Autophagy was decreased in those cells by both IL6 dependent and Fyn dependent mechanisms. Furthermore, in the denervated mouse model, not only both Fyn and IL6 gene expressions as well as the key muscle specific E3 ubiquitin ligases, Atrogin1 and MuRf1 were increased but the expression and phosphorylation levels of STAT3 were also augmented, while the autophagy activity was decreased. We believe that a denervated mouse model alone is not enough as a model for sarcopenia, thus we next introduced a hind limb suspension mouse model that promotes disuse atrophy by suspending the hind limb. Using this model, we found that muscle atrophy was observed mainly in the soleus muscle, tibialis anterior muscle, and the gastrocnemius muscle with Atrogin1 and MuRf1 increased. Increase of both IL6 and STAT3 expression/phosphorylation were also observed in the muscles of hind limb suspension mice. Autophagy activity, examined by intraperitoneal administration of colchicine, was decreased. These results strongly suggest that Fyn is involved not only in the metabolic syndrome but also in the pathogenesis of sarcopenia, and may lead to a better understanding of the pathology of sarcopenia obesity and the development of therapeutic methods.