Running improves muscle mass by activating autophagic flux and inhibiting ubiquitination degradation in mdx mice.

BACKGROUND: Exercise therapy can improve muscle mass, strengthen muscle and cardiorespiratory function, and may be an excellent adjunctive treatment option for Duchenne muscular dystrophy. METHODS: This article investigates the effects of 10 weeks of treadmill training on skeletal muscle in control and mdx mice. Hematoxylin and eosin (H&E) staining was used to detect the morphometry of skeletal muscle; the grip strength test, suspension test, and rotarod test were used to detect limb muscle strength of mice, and Aurora Scientific Instruments were used to detect in vivo Muscle Stimulation Measuring Maximum Force of pre-fatigue and post-fatigue. The expression levels of myogenic proteins, ubiquitination markers, autophagy pathway proteins, and the proportion of different muscle fiber types were detected. RESULTS: The experimental results show that running exercise can significantly improve the muscle mass of mdx mice, promote muscle strength, endurance, and anti-fatigue ability, reverse the pathological state of skeletal muscle destruction in mdx mice, and promote muscle regeneration. WB experiments showed that running inhibited the ubiquitination and degradation of muscle protein in mdx mice, inhibited AKT activation, decreased phosphorylated FoxO1 and FoxO3a, and restored the suppressed autophagic flux. Running enhances muscle strength and endurance by comprehensively promoting the expression of Myh1/2/4/7 fast and slow muscle fibers in mdx mice. CONCLUSIONS: Running can inhibit the degradation of muscle protein in mdx mice, and promote the reuse and accumulation of proteins, thereby slowing down muscle loss. Running improves skeletal muscle mass by activating autophagic flux and inhibiting ubiquitination degradation in mdx mice.

Roles of super enhancers and enhancer RNAs in skeletal muscle development and disease.

Skeletal muscle development is a multistep biological process regulated by a variety of myogenic regulatory factors, including MyoG, MyoD, Myf5, and Myf6 (also known as MRF4), as well as members of the FoxO subfamily. Differentiation and regeneration during skeletal muscle myogenesis contribute to the physiological function of muscles. Super enhancers (SEs) and enhancer RNAs (eRNAs) are involved in the regulation of development and diseases. Few studies have identified the roles of SEs and eRNAs in muscle development and pathophysiology. To develop approaches to enhance skeletal muscle mass and function, a more comprehensive understanding of the key processes underlying muscular diseases is needed. In this review, we summarize the roles of SEs and eRNAs in muscle development and disease through affecting of DNA methylation, FoxO subfamily, RAS-MEK signaling, chromatin modifications and accessibility, MyoD and cis regulating target genes. The summary could inform strategies to increase muscle mass and treat muscle-related diseases.

Nec-1 alleviated the deleterious effect of CoCl2 on C2C12 myoblast differentiation and fusion via the mTOR pathway.

Myoblast differentiation and fusion are vital for muscle development and repair in mammals. We previously showed that necrostatin-1(Nec-1) protects C2C12 myotubes from cobalt chloride (CoCl2)-induced pseudo-hypoxia. However, the function of Nec-1 in mouse C2C12 myoblast differentiation and fusion was still unknown. In this study, we found that CoCl2 substantially impaired C2C12 myoblast differentiation and fusion, and reduced the expression of Myh1, Myh2, Myh4, Myh7, myomaker, and myomerger. Nec-1 treatment rescued C2C12 myoblast differentiation and fusion and promoted the expression of myomaker and myomerger. Mechanistically, Nec-1 promoted C2C12 myoblast differentiation and fusion by inhibiting mTOR-mediated autophagy. Rapamycin abolished the increases in expression of muscle fusion-related genes, indicating that the upregulation of differentiation and fusion is mTOR-dependent. These results suggest that Nec-1 inhibited autophagy in an mTOR-dependent mechanism that is crucial for mTOR-induced C2C12 myoblast differentiation and fusion.

A new perspective on depression and neuroinflammation: Non-coding RNA.

The high incidence and relapse rate of depression, as well comorbidity with other diseases, has made depression one of the primary causes of years of life lived with disability. Moreover, the unknown biological mechanism of depression has made treatment difficult. Neuroinflammation is important in the pathogenesis of depression. Neuroinflammation may affect depression by regulating the production of immune factors, immune cell activation, neuron generation, synaptic plasticity, and neurotransmission. Non-coding RNAs (ncRNAs) may be a breakthrough link between depression and neuroinflammation, as ncRNAs participate in these biological changes. We summarize the functions and mechanisms of ncRNAs in neuroinflammation and depression, and predict ncRNAs that may regulate the occurrence and progression of depression through neuritis. These findings not only broaden our understanding of the genetic regulation of depression and neuroinflammation but also provide a new perspective of the underlying mechanism and aid in the design of novel prevention, diagnosis, and treatment strategies.

[Effects of Atrolnc-1 on immobilization induced muscular atrophy in mice hindlimbs].

Objective: To investigate the effects of Atrolnc-1 on immobilization induced muscular atrophy in mice hindlimbs. Methods: Male C57BL/6 mice were randomly divided into control group and immobilization group (n=10 per group). The control group did not receive any treatment. The right hindlimb of the Iimmobilization group was fixed by self-made plastic tube. After 2 weeks' immobilization, the gastrocnemius muscle was separated. Hematoxylin-eosin (HE) staining was used to observe the morphological changes and the cross-sectional area was calculated. The expressions of Atrogin-1 and atrophy-specific long non-coding RNA Atrolnc-1 were detected by quantitative real-time PCR (QRT-PCR). Western blot (WB) was used to detect the expressions of muscular atrophy fbox-1 protein (MAFbx/Atrogin-1), muscle ring finger1 (MuRF-1) in whole cell and phosphonated of nuclear factor kappaB (p-NF-κB) in cytoplasm and nucleus. Results: The gastrocnemius muscle was atrophy after 2 weeks' immobilization. Compared with the control group, the wet weight of gastrocnemius muscle was decreased (P＞0.05) and the permillage of wet weight/weight of gastrocnemius muscle was decreased significantly (P＜0.05). HE staining showed that the number of muscle fibers in the immobilization group were reduced, the muscle fibers were dissolved and arranged disorderly and the interstitial inflammatory cells were infiltrated; the cross-sectional area of muscle fibers was decreased (P＜0.01).The expression level of atrolnc-1 was increased in immobilization group (P＜0.01). The expression level of p-NF-κB in cytoplasm was decreased (P＜0.01), while the expression level of p-NF-κB was increased in nucleus ( P＜0.01). Besides, the expressions of atrogin-1 (P＜0.01) and MuRF-1 (P＜0.01) were increased. Conclusion: Immobilization induced gastrocnemius atrophy in mice may be related to the activation of NF-κB by Atrolnc-1 and then promote MuRF-1 expression.

Lnc-GD2H Promotes Proliferation by Forming a Feedback Loop With c-Myc and Enhances Differentiation Through Interacting With NACA to Upregulate Myog in C2C12 Myoblasts.

In the present study, the roles of a novel long non-coding RNA (lncRNA), lnc-GD2H, in promoting C2C12 myoblast proliferation and differentiation and muscle regeneration were investigated by quantitative polymerase chain reaction, western blotting, Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine (EdU), immunofluorescence staining, luciferase reporter, mass spectrometry, pulldown, chromatin immunoprecipitation, RNA immunoprecipitation assay, wound healing assays, and cardiotoxin (CTX)-induced muscle injury assays. It was observed that lnc-GD2H promoted myoblast proliferation as evidenced by the enhancement of the proliferation markers c-Myc, CDK2, CDK4, and CDK6, percentage of EdU-positive cells, and rate of cell survival during C2C12 myoblast proliferation. Additional experiments confirmed that c-Myc bound to the lnc-GD2H promoter and regulated its transcription. lnc-GD2H promoted cell differentiation with enhanced MyHC immunostaining as well as increased expression of the myogenic marker genes myogenin (Myog), Mef2a, and Mef2c during myoblast differentiation. Additional assays indicated that lnc-GD2H interacted with NACA which plays a role of transcriptional regulation in myoblast differentiation, and the enrichment of NACA at the Myog promoter was impaired by lnc-GD2H. Furthermore, inhibition of lnc-GD2H impaired muscle regeneration after CTX-induced injury in mice. lnc-GD2H facilitated the expression of proliferating marker genes and formed a feedback loop with c-Myc during myoblast proliferation. In differentiating myoblasts, lnc-GD2H interacted with NACA to relieve the inhibitory effect of NACA on Myog, facilitating Myog expression to promote differentiation. The results provide evidence for the role of lncRNAs in muscle regeneration and are useful for developing novel therapeutic targets for muscle disorders.

Knockdown of CNN3 Impairs Myoblast Proliferation, Differentiation, and Protein Synthesis via the mTOR Pathway.

BACKGROUND: Myogenesis is a complex process that requires optimal outside-in substrate-cell signaling. Calponin 3 (CNN3) plays an important role in regulating myogenic differentiation and muscle regeneration; however, the precise function of CNN3 in myogenesis regulation remains poorly understood. Here, we investigated the role of CNN3 in a knockdown model in the mouse muscle cell line C2C12. METHODS: Myoblast proliferation, migration, differentiation, fusion, and protein synthesis were examined in CNN3 knockdown C2C12 mouse muscle cells. Involvement of the mTOR pathway in CNN3 signaling was explored by treating cells with the mTOR activator MHY1485. The regulatory mechanisms of CNN3 in myogenesis were further examined by RNA sequencing and subsequent gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA). RESULTS: During proliferation, CNN3 knockdown caused a decrease in cell proliferation and migration. During differentiation, CNN3 knockdown inhibited myogenic differentiation, fusion, and protein synthesis in C2C12 cells via the AKT/mTOR and AMPK/mTOR pathways; this effect was reversed by MHY1485 treatment. Finally, KEGG and GSEA indicated that the NOD-like receptor signaling pathway is affected in CNN3 knockdown cell lines. CONCLUSION: CNN3 may promote C2C12 cell growth by regulating AKT/mTOR and AMPK/mTOR signaling. The KEGG and GSEA indicated that inhibiting CNN3 may activate several pathways, including the NOD-like receptor pathway and pathways involved in necroptosis, apoptosis, and inflammation.

Role of miRNAs and lncRNAs in dexamethasone-induced myotube atrophy in vitro.

Skeletal muscle atrophy is a well-known adverse effect of long-term glucocorticoid (GC) therapy. MicroRNAs (miRNAs or miRs) and long non-coding RNAs (lncRNAs) are important regulators in a number of physiological and pathological processes. However, the role of miRNAs and lncRNAs in the regulation of GC-treated muscle atrophy remains poorly understood. In the current study, muscular atrophy was induced and the results indicated that C2C12 myotubes were thinner than normal, while the expression of muscle ring finger protein 1 and Atrogin-1 was increased. The expression of nine miRNAs and seven lncRNAs associated with proliferation and differentiation were analyzed in a dexamethasone (DEX)-induced muscle atrophy cell model. In addition, the mRNA expression of the downstream targets of lncRNAs that were differentially expressed between DEX-treated and control cells were determined. The results indicated that the expression of miR-133a, miR-133b, miR-206 and five lncRNAs (increased Atrolnc-1, Dum, MAR1, linc-MD1 and decreased Myolinc) were significantly different between the DEX and the control group. Furthermore, the relative mRNA expression of Wnt5a and MyoD was significantly different between the two groups. The results of the current study indicated that some important miRNAs and lncRNAs are associated with DEX-induced muscle atrophy and have the potential to be further developed as a diagnostic tool for this condition.

Roles of lncRNAs and circRNAs in regulating skeletal muscle development.

The multistep biological process of myogenesis is regulated by a variety of myoblast regulators, such as myogenic differentiation antigen, myogenin, myogenic regulatory factor, myocyte enhancer factor2A-D and myosin heavy chain. Proliferation and differentiation during skeletal muscle myogenesis contribute to the physiological function of muscles. Certain non-coding RNAs, including long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), are involved in the regulation of muscle development, and the aberrant expressions of lncRNAs and circRNAs are associated with muscular diseases. In this review, we summarize the recent advances concerning the roles of lncRNAs and circRNAs in regulating the developmental aspects of myogenesis. These findings have remarkably broadened our understanding of the gene regulation mechanisms governing muscle proliferation and differentiation, which makes it more feasible to design novel preventive, diagnostic and therapeutic strategies for muscle disorders.

Expression patterns of regulatory lncRNAs and miRNAs in muscular atrophy models induced by starvation in vitro and in vivo.

Starvation or severe deprivation of nutrients, which is commonly seen in surgical patients, can result in catabolic changes in skeletal muscles, such as muscle atrophy. Therefore, it is important to elucidate the underlying molecular regulatory mechanisms during skeletal muscle atrophy. In the present study, muscular atrophy was induced by starvation and the results demonstrated that myosin heavy chain was decreased, whereas muscle RING finger protein 1 and atrogin­1 were increased, both in vitro and in vivo. The impact of starvation on the expression patterns of long non­coding RNAs (lncRNAs) and microRNAs (miRNAs) was next determined. The expression patterns of miR­23a, miR­206 and miR­27b in the starved mice exhibited similar trends as those in starved C2C12 cells in vitro, whereas the expression patterns of six other miRNAs (miR­18a, miR­133a, miR­133b, miR­186, miR­1a and miR­29b) differed between the in vivo and the in vitro starvation models. The present study indicated that in vitro expression of the selected miRNAs was not completely consistent with that in vivo. By contrast, lncRNAs showed excellent consistency in their expression patterns in both the in vitro and in vivo starvation models; six of the lncRNAs (Atrolnc­1, long intergenic non­protein coding RNA of muscle differentiation 1, Myolinc, lncRNA myogenic differentiation 1, Dum and muscle anabolic regulator 1) were significantly elevated in starved tissues and cells, while lnc­mg was significantly decreased, compared with the control groups. Thus, lncRNAs involved in muscle atrophy have the potential to be developed as diagnostic tools.

Differentially expressed coding and noncoding RNAs in CoCl2-induced cytotoxicity of C2C12 cells.

AIM: We aimed to explore potential regulators of coding and noncoding RNAs (ncRNAs) in Co(II) ion-induced myo cytotoxicity. MATERIALS & METHODS: We confirmed the toxic effects of Co(II) on mouse skeletal C2C12 myotubes by CoCl2, and performed the expression profiles of circular RNAs (circRNAs), long noncoding RNAs (lncRNAs) and mRNAs using microarray analysis. We constructed co-expression, competing endogenous RNA and cis/trans regulation networks for ncRNAs, and filtered 71 candidate circRNAs with coding potential. RESULTS: We identify 605 differentially expressed circRNAs, 4409 long noncoding RNAs and 3965 mRNAs. We also provided several ncRNAs regulation networks and presumed functions of circRNAs with coding potential. CONCLUSION: Our findings may reveal novel regulatory mechanisms underlying the noxious effects of CoCl2 in skeletal muscle.

Expression of circular RNAs during C2C12 myoblast differentiation and prediction of coding potential based on the number of open reading frames and N6-methyladenosine motifs.

The importance of circular RNAs (circRNAs) as regulators of muscle development and muscle-associated disorders is becoming increasingly apparent. To explore potential regulators of muscle differentiation, we determined the expression profiles of circRNAs of skeletal muscle C2C12 myoblasts and myotubes using microarray analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to explore circRNA functions. We also established competing endogenous RNA (ceRNA) networks using bioinformatics methods and predicted the coding potential of differentially expressed circRNAs. We found that 581 circRNAs were differentially regulated between C2C12 myoblasts and myotubes. Bioinformatics analysis suggested that the primary functions of the linear transcripts of the circRNAs were linked with organization of the cytoskeleton, calcium signaling, cell cycle, and metabolic pathways. ceRNA networks showed that the myogenic-specific genes myogenin, myocyte enhancer factor 2a, myosin heavy chain (Myh)-1, Myh7, and Myh7b could combine with 91 miRNAs and the top 30 upregulated circRNAs, forming 239 edges. According to the number of open reading frames and N6-methyladenosine motifs, we identified 224 circRNAs with coding potential, and performed GO and KEGG analyses based on the linear counterparts of 75 circRNAs. We determined that the 75 circRNAs were related to regulation of the actin cytoskeleton and metabolic pathways. We established expression profiles of circRNAs during C2C12 myoblast differentiation and predicted the function of differentially expressed circRNAs, which might be involved in skeletal muscle development. Our study offers new insight into the functions of circRNAs in skeletal muscle growth and development.

Necrostatin-1 protects C2C12 myotubes from CoCl2-induced hypoxia.

Necrostatin-1 (Nec-1) is a selective and potent allosteric inhibitor of necroptosis by specifically inhibiting the activity of receptor­interacting protein (RIP) 1 kinase. The aim of the present study was to determine the effect of Nec­1 on an anoxia model comprising mouse skeletal C2C12 myotubes. In the present study, a hypoxic mimetic reagent, cobalt chloride (CoCl2), was used to induce hypoxia in C2C12 myotubes. The cytotoxic effects of CoCl2­induced hypoxia were determined by a Cell Counting kit­8 assay and flow cytometry. Transmission electron microscopy (TEM) was used to characterize the morphological characteristics of dead cells at the ultrastructural level. To clarify the signaling pathways in CoCl2­mediated cell death, the expression levels of RIP1, RIP3, extracellular signal­regulated kinase (ERK)1/2, hypoxia­inducible factor (HIF)­1α and B cell lymphoma­2 adenovirus E1B 19­kDa interacting protein 3 (BNIP3) were investigated by western blotting. Oxidative stress was determined using 2',7'­dichlorofluorescin diacetate to measure intracellular reactive oxygen species (ROS) and the fluorescent dye JC­1 was used to measure mitochondrial membrane potential (Δψm). The results showed that the ratios of apoptotic and necrotic C2C12 cells were increased following CoCl2 treatment, typical necroptotic morphological characteristics were able to observe by TEM, whereas Nec­1 exhibited a protective effect against CoCl2­induced oxidative stress. Treatment with Nec­1 significantly decreased the levels of RIP1, p­ERK1/2, HIF­1α, BNIP3 and ROS induced by CoCl2, and promoted C2C12 differentiation. Nec­1 reversed the CoCl2­induced decrease in mitochondrial membrane potential. Together, these findings suggested that Nec­1 protected C2C12 myotubes under conditions of CoCl2-induced hypoxia.

Comprehensive analysis of lncRNAs and mRNAs with associated co-expression and ceRNA networks in C2C12 myoblasts and myotubes.

Long non-coding RNAs (lncRNAs) are emerging as important regulators in the modulation of muscle development and muscle-related diseases. To explore potential regulators of muscle differentiation, we determined the expression profiles of lncRNAs and mRNAs in C2C12 mouse myoblast cell line using microarray analysis. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to explore their function. We also constructed co-expression, cis/trans-regulation, and competing endogenous RNA (ceRNA) networks with bioinformatics methods. We found that 3067 lncRNAs and 3235 mRNAs were differentially regulated (fold change ≥2.0). Bioinformatics analysis indicated that the principal functions of the transcripts were related to muscle structure development and morphogenesis. Co-expression analysis showed 261 co-expression relationships between 233 lncRNAs and 10 mRNAs, and nine lncRNAs interacted with myog and MEF2C collectively. Cis/trans-regulation prediction revealed that lncRNA Myh6 could be a valuable gene via cis-regulation, and lncRNAs such as 2310043L19Ris, V00821, and AK139352 may participate in particular pathways regulated by transcription factors, including myog, myod1, and foxo1. The myog-specific ceRNA network covered 10 lncRNAs, 378 miRNAs, and 1960 edges. The upregulated lncRNAs Filip1, Myl1, and 2310043L19Rik may promote myog expression by acting as ceRNAs. Our results offer a new perspective on the modulation of lncRNAs in muscle differentiation.

Transcriptome analysis of CD34+ cells from myelodysplastic syndrome patients.

The myelodysplastic syndrome (MDS) represents a heterogeneous group of clonal hematologic stem cell disorders with the characteristic of ineffective hematopoiesis leading to low blood counts, and a risk of progression to acute myeloid leukemia (AML). To understand specific molecular characteristics of different MDS subtypes with del(5q), we analyzed the gene expression profiles of CD34+ cells from MDS patients of different databases and its enriched pathways. 44 genes, such as MME and RAG1, and eight related pathways were identified to be commonly changed, indicating their conserved roles in MDS development. Additionally, U43604 was identified to be specifically changed in three subtypes with del(5q), including refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS) and refractory anemia with excess blasts (RAEB). C10orf10 and CD79B were specifically changed in RA patients with del(5q), while POU2AF1 were in RARS patients with del(5q). We also analyzed specific pathways of MDS subtypes, such as "Glycosaminoglycan biosynthesis-chondroitin sulfate" which was specific identified in RARS patients. Importantly, those findings can be validated well using another MDS database. Taken together, our analysis identified specific genes and pathways associated with different MDS subtypes with del(5q).

Effects of Cobalt Chloride, a Hypoxia-Mimetic Agent, on Autophagy and Atrophy in Skeletal C2C12 Myotubes.

BACKGROUND: Hypoxia-induced autophagy and muscle wasting occur in several environmental and pathological conditions. However, the molecular mechanisms underlying the effects of the hypoxia-mimetic agent CoCl2 on autophagy and muscle atrophy are still unclear. METHODS: C2C12 myotubes were exposed to increasing concentrations of CoCl2 for 24 hours. Quantitative RT-PCR, Western blotting, and transmission electron microscopy were performed to confirm autophagy occurs. Autophagy proteins were measured to understand the molecule mechanisms. We also inhibited hypoxic autophagy and examined the changes in myogenin expression, myotubes formation, and apoptosis. RESULTS: Our results showed that CoCl2-mimicked hypoxia upregulated the expression of the autophagy-related proteins LC3, HIF-1α, BNIP3, p-AMPKα, and beclin-1, whereas p62 and p-mTOR were downregulated. In addition, the autophagosome could be observed after CoCl2 induction. The expression of the autophagy-related E3 ligase parkin and the muscle-specific ubiquitin ligase atrogin-1 was increased by CoCl2. Inhibition of autophagy by 3MA increased myogenin expression and promoted myotubes formation and the percentage of cell death was decreased. CONCLUSIONS: Our results confirmed that CoCl2-mimicked hypoxia induced autophagy via the HIF-1α/BNIP3/beclin-1 and AMPK/mTOR pathways. Our results also revealed an important link between autophagy and muscle atrophy under hypoxia, which may help to develop new therapeutic strategies for muscle diseases.