Herein we report that the 18-base telomeric oligodeoxynucleotides (ODNs) designed from the Lactobacillus rhamnosus GG genome promote differentiation of skeletal muscle myoblasts which are myogenic precursor cells. We termed these myogenetic ODNs (myoDNs). The activity of one of the myoDNs, iSN04, was independent of Toll-like receptors, but dependent on its conformational state. Molecular simulation and iSN04 mutants revealed stacking of the 13–15th guanines as a core structure for iSN04. The alkaloid berberine bound to the guanine stack and enhanced iSN04 activity, probably by stabilizing and optimizing iSN04 conformation. We further identified nucleolin as an iSN04-binding protein. Results showed that iSN04 antagonizes nucleolin, increases the levels of p53 protein translationally suppressed by nucleolin, and eventually induces myotube formation by modulating the expression of genes involved in myogenic differentiation and cell cycle arrest. This study shows that bacterial-derived myoDNs serve as aptamers and are potential nucleic acid drugs directly targeting myoblasts.

Myoblasts play a central role during skeletal muscle formation and growth. Precise understanding of myoblast properties is thus indispensable for meat production. Herein, we report the cellular characteristics and gene expression profiles of primary-cultured myoblasts of layer and broiler chickens. Broiler myoblasts actively proliferated and promptly differentiated into myotubes compared to layer myoblasts, which corresponds well with the muscle phenotype of broilers. Transcriptomes of layer and broiler myoblasts during differentiation were quantified by RNA sequencing. Ontology analyses of the differentially expressed genes (DEGs) provided a series of extracellular proteins as putative markers for characterization of chicken myogenic cells. Another ontology analyses demonstrated that broiler myogenic cells are rich in cell cycle factors and muscle components. Independent of these semantic studies, principal component analysis (PCA) statistically defined two gene sets: one governing myogenic differentiation and the other segregating layers and broilers. Thirteen candidate genes were identified with a combined study of the DEGs and PCA that potentially contribute to proliferation or differentiation of chicken myoblasts. We experimentally proved that one of the candidates, enkephalin, an opioid peptide, suppresses myoblast growth. Our results present a new perspective that the opioids present in feeds may influence muscle development of domestic animals.

Myoblasts are myogenic precursors that develop into myotubes during muscle formation. Improving efficiency of myoblast differentiation is important for advancing meat production by domestic animals. We recently identified novel oligodeoxynucleotides (ODNs) termed myogenetic ODNs (myoDNs) that promote the differentiation of mammalian myoblasts. An isoquinoline alkaloid, berberine, forms a complex with one of the myoDNs, iSN04, and enhances its activities. This study investigated the effects of myoDNs on chicken myoblasts to elucidate their species‐specific actions. Seven myoDNs (iSN01–iSN07) were found to facilitate the differentiation of chicken myoblasts into myosin heavy chain (MHC)‐positive myotubes. The iSN04–berberine complex exhibited a higher myogenetic activity than iSN04 alone, which was shown to enhance the differentiation of myoblasts into myotubes and the upregulation of myogenic gene expression (MyoD, myogenin, MHC, and myomaker). These data indicate that myoDNs promoting chicken myoblast differentiation may be used as potential feed additives in broiler diets.

Cancer cachexia is characterized by irreversible muscle loss which is a critical factor in the prognosis of cancer patients. Myoblasts are myogenic precursor cells that are required to maintain skeletal muscle tissue. Previous studies reported that cancer-released factors deteriorate myoblast differentiation, which is one of the causes of cachexia-associated muscle wasting. We recently identified the myogenetic oligodeoxynucleotide, iSN04, which serves as an anti-nucleolin aptamer and promotes myogenesis. The present study investigated the effects of iSN04 on human myoblasts exposed to a conditioned medium (CM) of cancer cells. CM of colon cancer cell lines LoVo and HCT-116 significantly impaired myogenic differentiation and the myotube formation of human myoblasts by inducing the expression of inflammatory cytokines such as interleukin (IL)-1β and tumor necrosis factor-α (TNF-α); however, the CM of the colon fibroblast cell line CCD-18Co did not. Intriguingly, iSN04 completely reversed the deterioration of myoblast differentiation by LoVo-CM by upregulating MyoD and myogenin, and downregulating myostatin, IL-1β, and TNF-α. TNF-α, of which a high level was produced in LoVo, alone inhibited myogenic differentiation and induced IL-1β, IL-6, and IL-8 transcriptions of myoblasts; however, pre-treatment with iSN04 reversed TNF-α-induced cachectic phenotypic features. The results indicate that iSN04 protects myoblasts against the effects of cancer-released factors and maintains their myogenic activity. This study provides a novel therapeutic strategy to prevent muscle loss associated with cancer cachexia.

Cell-cell fusion has been a great technology to generate valuable hybrid cells and organisms such as hybridomas. In this study, skeletal muscle myoblasts were utilized to establish a novel method for autonomous xenogenic cell fusion. Myoblasts are mononuclear myogenic precursor cells and fuse mutually to form multinuclear myotubes. We generated murine myoblasts (mMBs) expressing green fluorescent protein (GFP) termed mMB-GFP, and the chick myoblasts (chMBs) expressing Discosoma red fluorescent protein (DsRed) termed chMB-DsRed. mMB-GFP and chMB-DsRed were cocultured and induced to differentiate. After 24 h, the multinuclear myotubes expressing both GFP and DsRed were observed, indicating that mMBs and chMBs interspecifically fuse. These GFP /DsRed hybrid myotubes were able to survive and grew to hyper-multinucleated mature form. We also found that undifferentiated mMB-GFP efficiently fuse to the chMB-DsRed-derived myotubes. This is the first evidence for the autonomous xenogenic fusion of mammalian and avian cells. Myoblast-based fusogenic technique will open up an alternative direction to create novel hybrid products.