Muscle-specific Protein Research Articles

Effects of Physical Exercise on MuRF-1/TRIM63 mRNA Expression in Humans: A Systematic Review

Background/Objectives: Muscle-specific RING finger protein 1 (MuRF-1) is a pivotal regulator of muscle protein breakdown, an essential process for post-exercise muscle adaptation. This systematic review aimed to evaluate the effects of physical exercise on MuRF-1 mRNA expression in humans. Methods: A literature search was conducted in PubMed, Scopus, Cochrane Library, Google Scholar, and Web of Science following the PRISMA guidelines. The search was limited to studies published from 1 January 2001 to 1 December 2024. The inclusion and exclusion criteria were defined using the PICOS strategy. Two investigators independently performed the study selection, data extraction, and assessment of methodological quality, with any disagreements resolved by a third investigator. The PEDro scale was used to evaluate the risk of bias. Results: Forty-six studies met the eligibility criteria and were included. The findings evidenced that physical exercise significantly modulates MuRF-1 mRNA expression in humans. Resistance exercise induces transient increases, typically peaking between 1 and 4 h, whereas endurance exercise elicits similar responses within 40 min to 4 h post-exercise. Combined exercise protocols that include resistance and endurance exercises significantly increased MuRF-1 mRNA expression at 3 h post-exercise. The effects of physical exercise on MuRF-1 mRNA expression are influenced by factors such as exercise order, intensity, contraction mode, age, sex, and fitness level. Conclusions: This systematic review shows that MuRF-1 mRNA expression is significantly modulated by physical exercise in humans and is sensitive to different exercise modalities. These findings suggest that this key protein involved in muscle protein breakdown and turnover is essential for exercise-induced adaptations, contributing to skeletal muscle recovery and remodeling after exercise.

Recessive but damaging alleles of muscle-specific ribosomal protein gene RPL3L drive neonatal dilated cardiomyopathy.

The heart employs a specialized ribosome in its muscle cells to translate genetic information into proteins, a fundamental adaptation with an elusive physiological role. Its significance is underscored by the discovery of neonatal patients suffering from often fatal heart failure caused by rare compound heterozygous variants in RPL3L, a muscle-specific ribosomal protein that replaces the ubiquitous RPL3 in cardiac ribosomes. RPL3L -linked heart failure represents the only known human disease arising from mutations in tissue-specific ribosomes, yet the underlying pathogenetic mechanisms remain poorly understood despite an increasing number of reported cases. While the autosomal recessive inheritance pattern suggests a loss-of-function mechanism, Rpl3l -knockout mice display only mild phenotypes, attributed to up-regulation of the ubiquitous Rpl3. Interestingly, living human knockouts of RPL3L have been identified. Here, we report two new cases of RPL3L -linked severe neonatal heart failure and uncover an unusual pathogenetic mechanism through integrated analyses of population genetic data, patient cardiac tissue, and isogenic cells expressing RPL3L variants. Our findings demonstrate that patient hearts lack sufficient RPL3 compensation. Moreover, contrary to a simple loss-of-function mechanism often associated with autosomal recessive diseases, RPL3L -linked disease is driven by a combination of gain-of-toxicity and loss-of-function. Most patients carry a recurrent toxic missense variant alongside a non-recurrent loss-of-function variant. The non-recurrent variants trigger partial compensation of RPL3 similar to Rpl3l -knockout mice. In contrast, the recurrent missense variants exhibit increased affinity for the RPL3/RPL3L chaperone GRWD1 and 60S biogenesis factors, sequester 28S rRNA in the nucleus, disrupt ribosome biogenesis, and trigger severe cellular toxicity that extends beyond the loss of ribosomes. These findings elucidate the pathogenetic mechanisms underlying muscle-specific ribosome dysfunction in neonatal heart failure, providing critical insights for genetic screening and therapeutic development. Our findings also suggest that gain-of-toxicity mechanisms may be more widespread in autosomal recessive diseases, especially for those involving genes with paralogs.

Magnetic 3D bioprinting of skeletal muscle spheroid for a spheroid-based screening assay

Over the past decade, there has been a rapid development in the use of magnetic three dimensional (3D) based cell culture systems. Concerning the skeletal muscle, 3D culture systems can provide biological insights for translational clinical research in the fields of muscle physiology and metabolism. These systems can enhance the cell culture environment by improving spatially-oriented cellular assemblies and morphological features closely mimicking the in vivo tissues/organs, since they promote strong interactions between cells and the extracellular matrix (ECM). However, the time-consuming and complex nature of 3D traditional culture techniques pose a challenge to the widespread adoption of 3D systems. Herein, a bench protocol is presented for creating an innovative, promptly assembled and user-friendly culture platform for the magnetic 3D bioprinting of skeletal muscle spheroids. Our protocol findings revealed consistent morphological outcomes and the functional development of skeletal muscle tissue, as evidenced by the expression of muscle-specific contractile proteins and myotubes and the responsiveness to stimulation with cholinergic neurotransmitters. This proof-of-concept protocol confirmed the future potential for further validation and application of spheroid-based assays in human skeletal muscle research.

N6-Methyladenosine RNA Modification Regulates the Differential Muscle Development in Large White and Ningxiang Pigs.

N6-methyladenosine (m6A) is the most common modification in eukaryotic RNAs. Growing research indicates that m6A methylation is crucial for a multitude of biological processes. However, research on the m6A modifications in the regulation of porcine muscle growth is lacking. In this study, we identified differentially expressed genes in the neonatal period of muscle development between Large White (LW) and NingXiang (NX) pigs and further reported m6A methylation patterns via MeRIP-seq. We found that m6A modification regulates muscle cell development, myofibrils, cell cycle, and phosphatase regulator activity during the neonatal phase of muscle development. Interestingly, differentially expressed genes in LW and NX pigs were mainly enriched in pathways involved in protein synthesis. Furthermore, we performed a conjoint analysis of MeRIP-seq and RNA-seq data and identified 27 differentially expressed and m6A-modified genes. Notably, a typical muscle-specific envelope transmembrane protein, WFS1, was differentially regulated by m6A modifications in LW and NX pigs. We further revealed that the m6A modification accelerated the degradation of WFS1 in a YTHDF2-dependent manner. Noteworthy, we identified a single nucleotide polymorphism (C21551T) within the last exon of WFS1 that resulted in variable m6A methylation, contributing to the differing WFS1 expression levels observed in LW and NX pigs. Our study conducted a comprehensive analysis of the m6A modification on NX and LW pigs during the neonatal period of muscle development, and elucidated the mechanism by which m6A regulates the differential expression of WFS1 in the two breeds.

Effect of Isoflavone on Muscle Atrophy in Ovariectomized Mice.

Sarcopenia, characterized by muscle mass decline due to aging or other causes, is exacerbated by decreased estrogen levels after menopause in women. Isoflavones, a class of flavonoids acting on estrogen receptors, may have beneficial effects on metabolic disorders. We examined these effects in ovariectomized mice fed a high-fat, high-sucrose diet (HFHSD). At 7 weeks old, female C57BL6/J mice (18-20 g, n = 12) underwent bilateral ovariectomy (OVX), and were then fed a high-fat, high-sucrose diet starting at 8 weeks of age. Half of the mice received isoflavone water (0.1%). Metabolic analyses, including glucose and insulin tolerance tests, were conducted. Muscle analysis involved grip strength assays, next-generation sequencing, quantitative RT-PCR, and western blotting of skeletal muscle after euthanizing the mice at 14 weeks old. Additionally, 16S rRNA gene sequence analysis of the gut microbiota was performed. The results demonstrated that isoflavone administration did not affect body weight, glucose tolerance, or lipid metabolism. In contrast, isoflavone-treated mice had higher grip strength. Gene expression analysis of the soleus muscle revealed decreased Trim63 expression, and western blotting showed inactivation of muscle-specific RING finger protein 1 in isoflavone-treated mice. Gut microbiota analysis indicated higher Bacteroidetes and lower Firmicutes abundance in the isoflavone group, along with increased microbiota diversity. Gene sets related to TNF-α signaling via NF-κB and unfolded protein response were negatively associated with isoflavones. Isoflavone intake alters gut microbiota and increases muscle strength, suggesting a potential role in improving sarcopenia in menopausal women.

Fat body glycolysis defects inhibit mTOR and promote distant muscle disorganization through TNF-α/egr and ImpL2 signaling in Drosophila larvae

The fat body in Drosophila larvae functions as a reserve tissue and participates in the regulation of organismal growth and homeostasis through its endocrine activity. To better understand its role in growth coordination, we induced fat body atrophy by knocking down several key enzymes of the glycolytic pathway in adipose cells. Our results show that impairing the last steps of glycolysis leads to a drastic drop in adipose cell size and lipid droplet content, and downregulation of the mTOR pathway and REPTOR transcriptional activity. Strikingly, fat body atrophy results in the distant disorganization of body wall muscles and the release of muscle-specific proteins in the hemolymph. Furthermore, we showed that REPTOR activity is required for fat body atrophy downstream of glycolysis inhibition, and that the effect of fat body atrophy on muscles depends on the production of TNF-α/egr and of the insulin pathway inhibitor ImpL2.

Hydrogen sulfide inhibits skeletal muscle ageing by up-regulating autophagy through promoting deubiquitination of adenosine 5'-monophosphate (AMP)-activated protein kinase α1 via ubiquitin specific peptidase 5.

Hydrogen sulfide (H2S), the third gasotransmitter discovered, regulates a variety of physiological functions. Whether H2S alleviates skeletal muscle ageing by regulating autophagy has not been reported. Mice were administered 150mg/kg/day of D-galactose (D-gal), and C2C12 myotubes were cultured in 20g/L D-gal to induce ageing. Sodium hydrosulfide (NaHS) was employed as an exogenous donor in the treatment group. The intracellular concentration of H2S was quantified by the 7-azido-4-methylcoumarin fluorescence probe. The proteins involved in the ubiquitin-mediated degradation of AMPKα1 were detected by liquid chromatography tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation (Co-IP). S-sulfhydration of USP5 was tested by a biotin-switch assay. Associated proteins were analysed by western blot. NaHS was found to effectively restore the H2S content in both ageing gastrocnemius (+91.89%, P<0.001) and C2C12 myotubes (+27.55%, P<0.001). In comparison to theD-gal group, NaHS was observed to increase the mean cross-sectional area of muscle fibres (+44.91%, P<0.001), to decrease the collagen volume fraction of gastrocnemius (-81.32%, P=0.001) and to reduce the β-galactosidase-positive area of C2C12 myotubes (-28.74%, P<0.001). NaHS was also found to reverse the expression of muscle atrophy F box protein (MAFbx), muscle-specific RING finger protein 1 (MuRF1), Cyclin D1 and p21 in the ageing gastrocnemius tissue (MAFbx: -31.73%, P=0.008; MuRF1: -32.37%, P=0.003; Cyclin D1: +45.34%, P=0.010; p21: -25.53%, P=0.022) and C2C12 myotubes (MAFbx: -16.38%, P<0.001; MuRF1: -16.45%, P=0.003; Cyclin D1: +40.23%, P<0.001; p21: -35.85%, P=0.026). The AMPKα1-ULK1 pathway was activated and autophagy was up-regulated in NaHS-treated gastrocnemius tissue (p-AMPKα1: +61.61%, P=0.018; AMPKα1: +30.64%, P=0.010; p-ULK1/ULK1: +85.87%, P=0.005; p62: -29.07%, P<0.001; Beclin1: +24.75%, P=0.007; light chain 3 II/I [LC3 II/I]: +55.78%, P=0.004) and C2C12 myotubes (p-AMPKα1: +77.49%, P=0.018; AMPKα1: +26.18%, P=0.022; p-ULK1/ULK1: +38.34%, P=0.012; p62: -9.02%, P=0.014; Beclin1: +13.36%, P<0.001; LC3 II/I: +79.38%, P=0.017; autophagy flux: +24.88%, P=0.034) compared with the D-gal group. The effects of NaHS on autophagy were comparable to those of acadesine and LYN-1604, and chloroquine could reverse its effects on ageing. LC-MS/MS and Co-IP experiments demonstrated that USP5 is a deubiquitinating enzyme of AMPKα1. Following the knockdown of USP5, the activation of AMPKα1 was decreased (p-AMPKα1: -42.10%, P<0.001; AMPKα1: -43.93%, P<0.001), autophagy was inhibited (p-ULK1/ULK1: -27.51, P=0.001; p62: +36.00, P<0.001; Beclin1: -22.15%, P<0.001) and NaHS lost its ability to up-regulate autophagy. NaHS was observed to restore the expression (gastrocnemius: +62.17%, P<0.001; C2C12 myotubes: +37.51%, P=0.003) and S-sulfhydration (+53.07%, P=0.009) of USP5 and reduce the ubiquitination of AMPKα1. H2S promotes the deubiquitination of AMPKα1 by increasing the expression and S-sulfhydration of USP5, thereby up-regulating autophagy and alleviating skeletal muscle ageing.

CRISPR/Cas9-mediated knock-in of a fluorescent reporter into the target locus of interest in human pluripotent stem cells

The method presented herein is associated with the Lab Resource article titled “Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3, using CRISPR/CAS9-based gene targeting” [1]. The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is encoded by the human MYH6 gene, which is expressed in both the atria and ventricles during embryonic development and is predominantly expressed in the atria after birth [2]. Herein, the methods used to achieve CRISPR/SpCas9-mediated introduction of an EGFP reporter into αMHC, the target locus in human pluripotent stem cells (hPSCs) for cardiac lineage tracing and clinical cell sorting are described. The CRISPR-Cas9 system enables efficient replacement of the stop codon in the last exon of αMHC with a 2A non-joining peptide (T2A)-EGFP cassette. First, hPSCs are transfected with the donor construct and Cas9/sgRNA plasmids via electroporation and selected with neomycin for approximately 3 weeks. Thereafter, the established cell line exhibits typical characteristics of human embryonic stem cells (hESCs). When these cells differentiate into cardiomyocytes, the expression of EGFP is confirmed using confocal microscopy, flow cytometry analysis, and immunostaining.•The line enables monitoring of cell maturation events during human cardiac development.•The line is a valuable platform for cardiotoxicity tests and drug screening.•This method has already been employed in two original studies, as previously reported for reporter cell line generation using CRISPR/Cas9.

IGF1 promotes human myotube differentiation toward a mature metabolic and contractile phenotype.

Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal muscle models in vitro are incapable of fully recapitulating its physiological functions especially muscle contractility. By supplementation of insulin-like growth factor 1 (IGF1), a growth factor secreted by myofibers in vivo, we aimed to overcome these limitations. We monitored the differentiation process starting from primary human CD56-positive myoblasts in the presence/absence of IGF1 in serum-free medium in daily collected samples for 10 days. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon electrical pulse stimulation (EPS) following day 6. Myotubes without IGF1 were almost incapable of contraction. IGF1 treatment shifted the proteome toward skeletal muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7, and reduced MYH1/2 suggest a more oxidative phenotype further demonstrated by higher abundance of proteins of the respiratory chain and elevated mitochondrial respiration. IGF1-treatment also upregulated glucose transporter (GLUT)4 and increased insulin-dependent glucose uptake compared with myotubes differentiated without IGF1. To conclude, addition of IGF1 to serum-free medium significantly improves the differentiation of human myotubes that showed enhanced myofibril formation, response to electrical pulse stimulation, oxidative respiratory capacity, and glucose metabolism overcoming limitations of previous standards. This novel protocol enables investigation of muscular exercise on a molecular level.NEW & NOTEWORTHY Human skeletal muscle models are highly valuable to study how exercise prevents type 2 diabetes without invasive biopsies. Current models did not fully recapitulate the function of skeletal muscle especially during exercise. By supplementing insulin-like growth factor 1 (IGF1), the authors developed a functional human skeletal muscle model characterized by inducible contractility and increased oxidative and insulin-sensitive metabolism. The novel protocol overcomes the limitations of previous standards and enables investigation of exercise on a molecular level.

Role of P63 in Benign and Malignant Lesions of Breast

Introduction: Breast carcinoma is leading cause of cancer death in women. Breast lesions constitute heterogeneous group of diseases with wide variety of etiologies ranging from inflammatory-benign- malignant lesions. There are several reported markers for immunohistochemical detection of myoepithelial cells. Smooth muscle specific proteins, such as smooth muscle actin, smooth muscle myosin heavy chain, calponin and h-caldesmon are used to highlight myoepithelium. p63 antibody is myoepithelial cell marker that selectively stains nuclei. It is negative in stromal, myofibroblastic and adipocytic cells.Aims and Objectives: The aim of this study was to establish role of p63 expression in distinguishing benign breast lesions, premalignant lesions and malignant tumors of breast.Materials and Methods: 30 cases were selected from core biopsy, lumpectomy and mastectomy specimens ofbreast received at department of Pathology, Chalmeda Anand Rao Institute of Medical Sciences during the period of January 2021 to December 2021 and were studied prospectively. All specimens were processed according to CAP protocol and reported . Immunohistochemistry was performed to determine p63 expression in those specimens. p63 expression was evaluated as continuous positive/discontinuous positive/negative.Ethical Approval: This study was reviewed and approved by institute ethics committee, CAIMS, Karimnagar.Results: Among total 30 cases, 18 cases (60%) were benign lesions and all were positive for p63 expression. 3 cases (9.99%) were premalignant and were least positive for p63 expression. All malignant cases 9 cases (29.99%) were negative for p63 expression.Conclusion: The Positive correlation was seen between histomorphological features and p63 scoring in all the lesions, So p63 is good Immunohistochemical marker for evaluating breast lesions.

Potential of Lycii Radicis Cortex as an Ameliorative Agent for Skeletal Muscle Atrophy.

Lycii Radicis Cortex (LRC) is a traditional medicine in East Asia with various beneficial effects, including antioxidant, anti-inflammatory, anti-tumor, anti-diabetic, and anti-depressant properties. However, its potential effects on skeletal muscle atrophy have not been studied. In this study, the protective effects of LRC extract (LRCE) on dexamethasone (DEX)-induced muscle atrophy were investigated in C2C12 myotubes and mice. We evaluated the effect of LRCE on improving muscle atrophy using a variety of methods, including immunofluorescence staining, quantitative polymerase chain reaction (qPCR), Western blot, measurements of oxidative stress, apoptosis, ATP levels, and muscle tissue analysis. The results showed that LRCE improved myotube diameter, fusion index, superoxide dismutase (SOD) activity, mitochondrial content, ATP levels, expression of myogenin and myosin heavy chain (MHC), and reduced reactive oxygen species (ROS) production in dexamethasone-induced C2C12 myotubes. LRCE also enhanced protein synthesis and reduced protein degradation in the myotubes. In mice treated with DEX, LRCE restored calf thickness, decreased mRNA levels of muscle-specific RING finger protein 1 (MuRF1) and atrogin-1, and increased insulin-like growth factor 1 (IGF-1) mRNA level. Moreover, LRCE also repaired gastrocnemius muscle atrophy caused by DEX. Although human studies are not available, various preclinical studies have identified potential protective effects of LRCE against muscle atrophy, suggesting that it could be utilized in the prevention and treatment of muscle atrophy.

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.

Digenic inheritance involving a muscle-specific protein kinase and the giant titin protein causes a skeletal muscle myopathy

In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3−/−; ttn.1+/−) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases.

Myomerger Induces Membrane Hemifusion and Regulates Fusion Pore Expansion.

Membrane fusion is a crucial mechanism in a wide variety of important events in cell biology from viral infection to exocytosis. However, despite many efforts and much progress, cell-cell fusion has remained elusive to our understanding. Along the life of the fusion pore, large conformational changes take place from the initial lipid bilayer bending, passing through the hemifusion intermediates, and ending with the formation of the first nascent fusion pore. In this sense, computer simulations are an ideal technique for describing such complex lipid remodeling at the molecular level. In this work, we studied the role played by the muscle-specific membrane protein Myomerger during the formation of the fusion pore. We have conducted μs length atomistic and coarse-grained molecular dynamics, together with free-energy calculations using ad hoc collective variables. Our results show that Myomerger favors the hemifusion diaphragm-stalk transition, reduces the nucleation-expansion energy difference, and promotes the formation of nonenlarging fusion pores.

Mechanisms of RBM20 Cardiomyopathy: Insights From Model Systems.

RBM20 (RNA-binding motif protein 20) is a vertebrate- and muscle-specific RNA-binding protein that belongs to the serine-arginine-rich family of splicing factors. The RBM20 gene was first identified as a dilated cardiomyopathy-linked gene over a decade ago. Early studies in Rbm20 knockout rodents implicated disrupted splicing of RBM20 target genes as a causative mechanism. Clinical studies show that pathogenic variants in RBM20 are linked to aggressive dilated cardiomyopathy with early onset heart failure and high mortality. Subsequent studies employing pathogenic variant knock-in animal models revealed that variants in a specific portion of the arginine-serine-rich domain in RBM20 not only disrupt splicing but also hinder nucleocytoplasmic transport and lead to the formation of RBM20 biomolecular condensates in the sarcoplasm. Conversely, mice harboring a disease-associated variant in the RRM (RNA recognition motif) do not show evidence of adverse remodeling or exhibit sudden death despite disrupted splicing of RBM20 target genes. Thus, whether disrupted splicing, biomolecular condensates, or both contribute to dilated cardiomyopathy is under debate. Beyond this, additional questions remain, such as whether there is sexual dimorphism in the presentation of RBM20 cardiomyopathy. What are the clinical features of RBM20 cardiomyopathy and why do some individuals develop more severe disease than others? In this review, we summarize the reported observations and discuss potential mechanisms of RBM20 cardiomyopathy derived from studies employing in vivo animal models and in vitro human-induced pluripotent stem cell-derived cardiomyocytes. Potential therapeutic strategies to treat RBM20 cardiomyopathy are also discussed.

Uncovering the mechanisms of MuRF1-induced ubiquitylation and revealing similarities with MuRF2 and MuRF3

MuRF1 (Muscle-specific RING finger protein 1; gene name TRIM63) is a ubiquitin E3 ligase, associated with the progression of muscle atrophy. As a RING (Really Interesting New Gene) type E3 ligase, its unique activity of ubiquitylation is driven by a specific interaction with a UBE2 (ubiquitin conjugating enzyme). Our understanding of MuRF1 function remains unclear as candidate UBE2s have not been fully elucidated. In the present study, we screened human ubiquitin dependent UBE2s in vitro and found that MuRF1 engages in ubiquitylation with UBE2D, UBE2E, UBE2N/V families and UBE2W. MuRF1 can cause mono-ubiquitylation, K48- and K63-linked polyubiquitin chains in a UBE2 dependent manner. Moreover, we identified a two-step UBE2 dependent mechanism whereby MuRF1 is monoubiquitylated by UBE2W which acts as an anchor for UBE2N/V to generate polyubiquitin chains. With the in vitro ubiquitylation assay, we also found that MuRF2 and MuRF3 not only share the same UBE2 partners as MuRF1 but can also directly ubiquitylate the same substrates: Titin (A168-A170), Desmin, and MYLPF (Myosin Light Chain, Phosphorylatable, Fast Skeletal Muscle; also called Myosin Light Regulatory Chain 2). In summary, our work presents new insights into the mechanisms that underpin MuRF1 activity and reveals overlap in MuRF-induced ubiquitylation which could explain their partial redundancy in vivo.

Endurance Training and MitoQ Supplementation Increase PERM1 and SMYD1 Gene Expression and Improve Hemodynamic Function in the Cardiac Muscle of Male Wistar Rats

Background: Histone methyltransferase (SMYD1) and the muscle-specific protein PERM1 play an important role in maintaining cardiac energetics and function. The present study aimed to examine the effect of two types of endurance training (running and swimming) plus MitoQ supplementation on PERM1 and SMYD1 gene expression and the hemodynamic function of the cardiac muscle of male Wistar rats. Methods: The animals underwent MitoQ supplementation alone, endurance training alone, or endurance training+MitoQ supplementation for eight weeks. The gene expression of PERM1 and SMYD1, which was measured by real-time polymerase chain reaction (PCR) and quantified by 2-∆∆CT method, and hemodynamic function were compared between groups using two-way multivariate analysis of variance. Results: Cardiac gene expression of PERM1 and SMYD1 increased significantly through running (PERM1, P&lt;0.05; SMYD1, P&lt;0.01), swimming (PERM1, P&lt;0.05; SMYD1, P&lt;0.05) and MitoQ supplementation (PERM1, P&lt;0.001). The effects of MitoQ supplementation on the cardiac gene expression of PERM1 were additive to the effects of both running and swimming (PERM1, P&lt;0.001; SMYD1, P&lt;0.05). Swimming-induced enhancement in cardiac expression of PERM1 and SMYD1 was associated with a significant increase in±dP/dt max (P&lt;0.05). Conclusion: The expression of genes involved in cardiac metabolism can be affected by endurance training and this effect could be improved through MitoQ supplementation.

Rieske Iron-Sulfur Protein Mediates Pulmonary Hypertension Following Nicotine/Hypoxia Coexposure.

The high mortality rate in patients with chronic obstructive pulmonary disease (COPD) may be due to pulmonary hypertension (PH). These diseases are highly associated with cigarette smoke and its key component nicotine. Here, we created a novel animal model of PH using coexposure to nicotine (or cigarette smoke) and hypoxia. This heretofore unreported model showed significant early-onset pulmonary vasoremodeling and PH. Using newly generated mice with complementary smooth muscle-specific Rieske iron-sulfur protein (RISP) gene knockout and overexpression, we demonstrate that RISP is critically involved in promoting pulmonary vasoremodeling and PH, which are implemented by oxidative ataxia telangiectasia-mutated-mediated DNA damage and NF-κB-dependent inflammation in a reciprocal positive mechanism. Together, our findings establish for the first time an animal model of hypoxia-induced early-onset PH in which mitochondrial RISP-dependent DNA damage and NF-κB inflammation play critical roles in vasoremodeling. Specific therapeutic targets for RISP and related oxidative stress-associated signaling pathways may create unique and effective treatments for PH, chronic obstructive pulmonary disease, and their complications.

Culture Method and Regulation Mechanisms of Stages of C2C12 Cell Line Myogenesis

INTRODUCTION: C2C12 is an immortalized mouse myoblast cell line actively used as in vitro experimental models to study skeletal muscle metabolism in biomedical research. C2C12 cells can differentiate into myocytes under appropriate culture conditions. It is important to understand the specifics of each stage of myogenesis and its regulatory mechanisms for the targeted exposure and development of medical drugs, taking into account the mechanisms of pathogenesis of various diseases.&#x0D; AIM: To describe the method of culturing С2С12 cell line and to study regulatory mechanisms of С2С12 cell line myogenesis.&#x0D; MATERIALS AND METHODS: The study was conducted on C2C12 cell line. Differentiation of myoblasts was induced in the nutrient medium containing 2% horse serum. The differentiation stages were evaluated by studying the cells on the 1st, 4th and 7th days. Cells before differentiation were used as a comparison group. At each differentiation stage, the myoblast fusion index (IM) was evaluated with preliminary staining of the cells by Romanowsky–Giemsa method. The cell differentiation mechanism was evaluated by the level of myosin, a-actin, myogenic differentiation protein (MyoD), myogenin (MyoG) using Western blot method.&#x0D; RESULTS: On the 1st day of C2C12 cells culturing, formation of myotubes was observed (IM = 0.15 ± 0.05), on the 4th day — fusion of myoblasts with the formation of binucleated cells accompanied by an increase in the amount of MyoD and α-actin protein (IM = 0.44 ± 0.14). By the 7th day of differentiation, the fusion of cells increased with the formation of myotubes containing more than two nuclei; the content of MyoD and α-actin did not differ from the control, and the amount of MyoG and myosin increased (IM = 0.77 ± 0.04).&#x0D; CONCLUSION: The described method of culturing C2C12 cell line in Dulbecco’s Modified Eagle’s Medium with a high glucose content (4500 mg/l), containing 2% horse serum, L-glutamine (4 mМ), 100 Un/ml and 100 µg/ml of penicillin and streptomycin, is suitable for the formation of muscle cells, where MyoD and MyoG participate in the regulation of the increase in the amount of specific muscle proteins — α-actin and myosin.

Effects of electroacupuncture at "Jiaji" (EX-B 2) combined with neurodynamic mobilization on gastrocnemius muscle atrophy and expression of NF-κB and MuRF1 in rabbits after sciatic nerve injury.

To observe the effects of electroacupuncture (EA) at "Jiaji" (EX-B 2) combined with neurodynamic mobilization (NM) on the cross-sectional area of the gastrocnemius muscle fibers after sciatic nerve injury in rabbits, and the expression of nuclear factor κB (NF-κB) and muscle-specific ring-finger protein 1 (MuRF1). A total of 180 common-grade New Zealand rabbits (half male and half female) were randomly divided into five groups, i.e. a normal control group, a model control group, a NM group, an EA group and a combined intervention group, 36 rabbits in each group. Except in the normal control group, clipping method was used to prepare the model of sciatic nerve injury in the rest groups. On the 3rd day of successful modeling, NM was delivered in the NM group. In the EA group, EA was exerted at bilateral "Jiaji" (EX-B 2) of L4 to L6, stimulated with disperse-dense wave and the frequency of 2 Hz/100 Hz. In the combined intervention group, after EA delivered at bilateral "Jiaji" (EX-B 2) of L4 to L6 , NM was operated. The intervention in each group was delivered once daily, for 6 days a week, and lasted 1, 2 or 4 weeks according to the collection time of sample tissue. After 1, 2 and 4 weeks of intervention, in each group, the toe tension reflex score and the modified Tarlov test score were observed; the morphology of the gastrocnemius muscle was observed by HE staining and the cross-sectional area of muscular fiber was measured; using Western blot method, the expression of NF-κB and MuRF1 of the gastrocnemius muscle was detected. After 1, 2 and 4 weeks of intervention, the toe tension reflex scores and the modified Tarlov scores in the model control group were lower than those of the normal control group (P<0.05), and these two scores in the NM group, the EA group and the combined intervention group were all higher than those of the model control group (P<0.05); the scores in the combined intervention group were higher than those in the EA group and the NM group (P<0.05). The gastrocnemius fibers were well arranged and the myocyte morphology was normal in the normal control group. In the model control group, the gastrocnemius fibers were disarranged, the myocytes were irregular in morphology and the inflammatory cells were infiltrated in the local. In the NM group, the EA group and the combined intervention group, the muscle fibers were regularly arranged when compared with the model control group. After 1, 2 and 4 weeks of intervention, the cross-sectional areas of the gastrocnemius muscle fibers in the model control group were smaller than those of the normal control group (P<0.05). The cross-sectional areas in the NM group, the EA group and the combined intervention group were larger than those of the model control group (P<0.05), and the cross-sectional areas in the combined intervention group were larger than those in the NM group and the EA group (P<0.05). After intervention for 1, 2 and 4 weeks, the protein expressions of NF-κB and MuRF1 in the gastrocnemius muscle were higher in the model control group in comparison of those in the normal control group (P<0.05). In the NM group, the EA group and the combined intervention group, the expressions of NF-κB after intervention for 1, 2 and 4 weeks and the expressions of MuRF1 after 2 and 4 weeks of intervention were lower when compared with those in the model control group (P<0.05). In the combined intervention group, the protein expressions of NF-κB after intervention for 1, 2 and 4 weeks and the expressions of MuRF1 after 2 and 4 weeks of intervention were decreased when compared with those in the NM group and the EA group (P<0.05). Electroacupuncture at "Jiaji" (EX-B 2) combined with NM may increase the muscle strength and sciatic function and alleviate gastrocnemius muscle atrophy in the rabbits with sciatic nerve injury. The underlying mechanism is related to the inhibition of NF-κB and MuRF1 expression.