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.

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 N-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 (Nec-1) is a selective and potent allosteric inhibitor of necroptosis by specifically inhibiting the activity of receptor-interacting protein (RIP) 1 kinase. The aim of the present study was to determine the effect of Nec-1 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 CoCl2-induced hypoxia were determined by a Cell Counting kit-8 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 CoCl2-mediated cell death, the expression levels of RIP1, RIP3, extracellular signal-regulated kinase (ERK)1/2, hypoxia-inducible factor (HIF)-1α and B cell lymphoma-2 adenovirus E1B 19-kDa 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 JC-1 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 Nec-1 exhibited a protective effect against CoCl2-induced oxidative stress. Treatment with Nec-1 significantly decreased the levels of RIP1, p-ERK1/2, HIF-1α, BNIP3 and ROS induced by CoCl2, and promoted C2C12 differentiation. Nec-1 reversed the CoCl2-induced decrease in mitochondrial membrane potential. Together, these findings suggested that Nec-1 protected C2C12 myotubes under conditions of CoCl2-induced hypoxia.

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. K E Y W O R D ScircRNAs, development, differentiation, lncRNAs, proliferation, skeletal muscle

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