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.

Skeletal muscle atrophy is a common complication of cachexia, characterized by progressive bodyweight loss and decreased muscle strength, and it significantly increases the risks of morbidity and mortality in the population with atrophy. Numerous complications associated with decreased muscle function can activate catabolism, reduce anabolism, and impair muscle regeneration, leading to muscle wasting. microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), types of non-coding RNAs, are important for regulation of skeletal muscle development. Few studies have specifically identified the roles of miRNAs and lncRNAs in cellular or animal models of muscular atrophy during cachexia, and the pathogenesis of skeletal muscle wasting in cachexia is not entirely understood. To develop potential approaches to improve skeletal muscle mass, strength, and function, a more comprehensive understanding of the known key pathophysiological processes leading to muscular atrophy is needed. In this review, we summarize the known miRNAs, lncRNAs, and corresponding signaling pathways involved in regulating skeletal muscle atrophy in cachexia and other diseases. A comprehensive understanding of the functions and mechanisms of miRNAs and lncRNAs during skeletal muscle wasting in cachexia and other diseases will, therefore, promote therapeutic treatments for muscle atrophy.

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 atrogin-1 were increased, both in vitro and in vivo. The impact of starvation on the expression patterns of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) was next determined. The expression patterns of miR-23a, miR-206 and miR-27b in the starved mice exhibited similar trends as those in starved C2C12 cells in vitro, whereas the expression patterns of six other miRNAs (miR-18a, miR-133a, miR-133b, miR-186, miR-1a and miR-29b) 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 (Atrolnc-1, long intergenic non-protein 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 lnc-mg was significantly decreased, compared with the control groups. Thus, lncRNAs involved in muscle atrophy have the potential to be developed as diagnostic tools.