Guanidinoacetic acid (GAA), an amino acid derivative that is endogenous to animal tissues including muscle and nerve, has been reported to enhance muscular performance. MicroRNA (miRNA) is a post-transcriptional regulator that plays a key role in nutrient-mediated myogenesis. However, the effects of GAA on myogenic differentiation and skeletal muscle growth, and the potential regulatory mechanisms of miRNA in these processes have not been elucidated. In this study, we investigated the effects of GAA on proliferation, differentiation, and growth in C2C12 cells and mice. The results showed that GAA markedly inhibited the proliferation of myoblasts, along with the down-regulation of cyclin D1 (CCND1) and cyclin dependent kinase 4 (CDK4) mRNA expression, and the upregulation of cyclin dependent kinase inhibitor 1A (P21) mRNA expression. We also demonstrated that GAA treatment stimulated myogenic differentiation 1 (MyoD) and myogenin (MyoG) mRNA expression, resulting in an increase in the myotube fusion rate. Meanwhile, GAA supplementation promoted myotube growth through increase in total myosin heavy chain (MyHC) protein level, myotubes thickness and gastrocnemius muscle cross-sectional area. Furthermore, small RNA sequencing revealed that a total of eight miRNAs, including miR-133a-3p and miR-1a-3p cluster, showed differential expression after GAA supplementation. To further study the function of miR-133a-3p and miR-1a-3p in GAA-induced skeletal muscle growth, we transfected miR-133a-3p and miR-1a-3p mimics into myotube, which also induced muscle growth. Through bioinformatics and a dual-luciferase reporter system, the target genes of miR-133a-3p and miR-1a-3p were determined. These two miRNAs were shown to modulate the Akt/mTOR/S6K signaling pathway by restraining target gene expression. Taken together, these findings suggest that GAA supplementation can promote myoblast differentiation and skeletal muscle growth through miR-133a-3p- and miR-1a-3p-induced activation of the AKT/mTOR/S6K signaling pathway.

Recent evidence suggests that hypoxia caused by acute myocardial infarction can induce cardiomyocyte apoptosis. Exosomes are signalling mediators that contribute to intercellular communication by transporting cytosolic components including miRNAs, mRNAs, and proteins. However, the systemic regulation and function of exosomal miRNAs in hypoxic cardiomyocytes are currently not well understood. Here, we used small RNA sequencing to investigate the effects of hypoxia stress on miRNAome of rat cardiomyoblast cells (H9c2) and corresponding exosomes. We identified 92 and 62 miRNAs in cells and exosomes, respectively, that were differentially expressed between hypoxia and normoxia. Hypoxia strongly modulated expression of hypoxia-associated miRNAs in H9c2 cells, and altered the miRNAome of H9c2 cells-derived exosomes. Functional enrichment analysis revealed extensive roles of differentially expressed exosomal miRNAs in the HIF-1 signalling pathway and in apoptosis-related pathways including the TNF, MAPK, and mTOR pathways. Furthermore, gain- and loss-of-function analysis demonstrated potential anti-apoptotic effects of the hypoxia-induced exosomal miRNAs, including miR-21-5p, miR-378-3p, miR-152-3p, and let-7i-5p; luciferase reporter assay confirmed that Atg12 and Faslg are targets of miR-152-3p and let-7i-5p, respectively. To summarize, this study revealed that hypoxia-induced exosomes derived from H9c2 cells loaded cardioprotective miRNAs, which mitigate hypoxia-induced H9c2 cells apoptosis.

Recent evidence suggests that testosterone deficiency can dramatically decrease the quality of sperm. MicroRNAs (miRNAs) are conserved mediators of post-transcriptional gene regulation in eukaryotes. However, the systemic regulation and function of miRNAs in sperm quality decline induced by testosterone deficiency has not been investigated. Here, we found that the sperm apoptosis was significantly enhanced and the sperm motility was dramatically decreased in hemicastrated pigs. We then used small RNA sequencing to detect miRNA profiles of sperm from pigs with prepubertal hemicastration (HC) and compared them with control libraries. We identified 16 differentially expressed (DE) miRNAs between the sperm of prepubertal HC and control (CT) pigs. Functional enrichment analysis indicated that the target genes of these DE miRNAs were mainly enriched in apoptosis-related pathways including the p53, mitogen-activated protein kinase (MAPK), and mammalian target of rapamycin (mTOR) pathways. Furthermore, gain- and loss-of-function analyses demonstrated potential anti-apoptotic effects of the DE miRNAs miR-26a-5p and let-7g-5p on sperm cells. The luciferase reporter assay confirmed that PTEN and PMAIP1 are targets of miR-26a-5p and let-7g-5p, respectively. Spearman’s correlation analysis revealed significantly positive correlations between the sperm and its corresponding seminal plasma exosomes regarding the miRNA expression levels. In conclusion, testosterone deficiency-induced changes in the miRNA components of seminal plasma exosomes secreted by the genital tract may partially elucidate sperm miRNAome alterations, which are further responsible for the decline of sperm motility.

Acute myocardial infarction (AMI) is an ischemic heart disease with high mortality worldwide. AMI triggers a hypoxic microenvironment and induces extensive myocardial injury, including autophagy and apoptosis. MiRNAs, which are a class of posttranscriptional regulators, have been shown to be involved in the development of ischemic heart diseases. We have previously reported that hypoxia significantly alters the miRNA transcriptome in rat cardiomyoblast cells (H9c2), including miR-27a-5p. In the present study, we further investigated the potential function of miR-27a-5p in the cardiomyocyte response to hypoxia, and showed that miR-27a-5p expression was downregulated in the H9c2 cells at different hypoxia-exposed timepoints and the myocardium of a rat AMI model. Follow-up experiments revealed that miR-27a-5p attenuated hypoxia-induced cardiomyocyte injury by regulating autophagy and apoptosis via Atg7, which partly elucidated the anti-hypoxic injury effects of miR-27a-5p. Taken together, this study shows that miR-27a-5p has a cardioprotective effect on hypoxia-induced H9c2 cell injury, suggesting it may be a novel target for the treatment of hypoxia-related heart diseases.

The normal growth and development of skeletal muscle is essential for the health of the body. The regulation of skeletal muscle by intestinal microorganisms and their metabolites has been continuously demonstrated. Acetate is the predominant short-chain fatty acids synthesized by gut microbiota through the fermentation of dietary fiber; however, the underlying molecular mechanisms governing the interaction between acetate and skeletal muscle during the rapid growth stage remains to be further elucidated. Herein, specific pathogen-free (SPF) mice, germ-free (GF) mice, and germ-free mice supplemented with sodium acetate (GS) were used to evaluate the effects of acetate on the skeletal muscle growth and development of young mice with gut microbiota deficiency. We found that the concentration of serum acetate, body mass gain, succinate dehydrogenase activity, and expression of the myogenesis maker gene of skeletal muscle in the GS group were higher than those in the GF group, following sodium acetate supplementation. Furthermore, the transcriptome analysis revealed that acetate activated the biological processes that regulate skeletal muscle growth and development in the GF group, which are otherwise inhibited due to a gut microbiota deficiency. The in vitro experiment showed that acetate up-regulated Gm16062 to promote skeletal muscle cell differentiation. Overall, our findings proved that acetate promotes skeletal muscle growth and development in young mice via increasing Gm16062 expression.

Skeletal muscle formation is an extremely important step in animal growth and development. Recent studies have found that TMEM8c (also known as Myomaker, MYMK), a muscle-specific transmembrane protein, can promote myoblast fusion and plays a key role in the normal development of skeletal muscle. However, the effect of Myomaker on porcine (Sus scrofa) myoblast fusion and the underlying regulatory mechanisms remain largely unknown. Therefore, in this study, we focused on the role and corresponding regulatory mechanism of the Myomaker gene during skeletal muscle development, cell differentiation, and muscle injury repair in pigs. We obtained the entire 3′ UTR sequence of porcine Myomaker using the 3′ RACE approach and found that miR-205 inhibited porcine myoblast fusion by targeting the 3′ UTR of Myomaker. In addition, based on a constructed porcine acute muscle injury model, we discovered that both the mRNA and protein expression of Myomaker were activated in the injured muscle, while miR-205 expression was significantly inhibited during skeletal muscle regeneration. The negative regulatory relationship between miR-205 and Myomaker was further confirmed in vivo. Taken together, the present study reveals that Myomaker plays a role during porcine myoblast fusion and skeletal muscle regeneration and demonstrates that miR-205 inhibits myoblast fusion through targeted regulation of the expression of Myomaker.