Age-Related Exosomal and Endogenous Expression Patterns of miR-1, miR-133a, miR-133b, and miR-206 in Skeletal Muscles

Skeletal muscle growth and maintenance depend on two tightly regulated processes, myogenesis and muscle regeneration. Both processes involve a series of crucial regulatory molecules including muscle-specific microRNAs, known as myomiRs. We recently showed that four myomiRs, miR-1, miR-133a, miR-133b, and miR-206, are encapsulated within muscle-derived exosomes and participate in local skeletal muscle communication. Although these four myomiRs have been extensively studied for their function in muscles, no information exists regarding their endogenous and exosomal levels across age. Here we aimed to identify any age-related changes in the endogenous and muscle-derived exosomal myomiR levels during acute skeletal muscle growth. The four endogenous and muscle-derived myomiRs were investigated in five skeletal muscles (extensor digitorum longus, soleus, tibialis anterior, gastrocnemius, and quadriceps) of 2-week–1-year-old wild-type male mice. The expression of miR-1, miR-133a, and miR-133b was found to increase rapidly until adolescence in all skeletal muscles, whereas during adulthood it remained relatively stable. By contrast, endogenous miR-206 levels were observed to decrease with age in all muscles, except for soleus. Differential expression of the four myomiRs is also inversely reflected on the production of two protein targets; serum response factor and connexin 43. Muscle-derived exosomal miR-1, miR-133a, and miR-133b levels were found to increase until the early adolescence, before reaching a plateau phase. Soleus was found to be the only skeletal muscle to release exosomes enriched in miR-206. In this study, we showed for the first time an in-depth longitudinal analysis of the endogenous and exosomal levels of the four myomiRs during skeletal muscle development. We showed that the endogenous expression and extracellular secretion of the four myomiRs are associated to the function and size of skeletal muscles as the mice age. Overall, our findings provide new insights for the myomiRs’ significant role in the first year of life in mice.

Dynamic transcriptome profiles of postnatal porcine skeletal muscle growth and development

Background Skeletal muscle growth and development are closely associated with the quantity and quality of pork production. We performed a transcriptomic analysis of 12 Longissimus dorsi muscle samples from Tibetan piglets at four postnatal stages of 0, 14, 30, and 60 days using RNA sequencing. Results According to the pairwise comparisons between the libraries of the muscle samples at the four postnatal stages, a total of 4115 differentially expressed genes (DEGs) were identified in terms of |log2(fold change)| ≥ 1 and an adjusted P value < 0.01. Short-time series expression miner (STEM) analysis of the DEGs identified eight significantly different expression profiles, which were divided into two clusters based on the expression pattern. DEGs in cluster I displayed a pattern of decreasing to a nadir, and then a rise, and the significantly enriched gene ontology (GO) terms detected using them were involved in multiple processes, of which the cell cycle, immunocyte activation and proliferation, as well as actin cytoskeleton organization, were the top three overrepresented processes based on the GO terms functional classification. DEGs in cluster II displayed a pattern of increasing to a peak, then declining, which mainly contributed to protein metabolism. Furthermore, besides the pathways related to immune system, a few diseases, and protein metabolism, the DEGs in clusters I and II were significantly enriched in pathways related to muscle growth and development, such as the Rap1, PI3K-Akt, AMPK, and mTOR signaling pathways. Conclusions This study revealed GO terms and pathways that could affect the postnatal muscle growth and development in piglets. In addition, this study provides crucial information concerning the molecular mechanisms of muscle growth and development as well as an overview of the piglet transcriptome dynamics throughout the postnatal period in terms of growth and development.

Anemic Hypoxemia Reduces Myoblast Proliferation and Muscle Growth in Late Gestation Fetal Sheep

Fetal skeletal muscle growth requires myoblast proliferation, differentiation, and fusion into myofibers in addition to protein accretion for fiber hypertrophy. Oxygen is an important regulator of this process. Therefore, we hypothesized that fetal anemic hypoxemia would inhibit skeletal muscle growth. Studies were performed in late gestation fetal sheep that were bled to anemic, and therefore hypoxemic, conditions beginning at ~125 days of gestation (term = 148 days) for 9 ± 0 days (n=19) and compared to control fetuses (n=16). A metabolic study was performed on gestational day ~134 to measure fetal protein kinetic rates. Myoblast proliferation and myofiber area were determined in biceps femoris (BF), tibialis anterior (TA), and flexor digitorum superficialis (FDS) muscles. mRNA expression of muscle regulatory factors was determined in BF. Fetal arterial hematocrit and oxygen content were 28% and 52% lower, respectively, in anemic fetuses. Fetal weight and whole-body protein synthesis, breakdown, and accretion rates were not different between groups. Hindlimb length, however, was 7% shorter in anemic fetuses. TA and FDS muscles weighed less and FDS myofiber area was smaller in anemic fetuses compared to controls. The percentage of Pax7+ myoblasts that expressed Ki67 was lower in BF and tended to be lower in FDS from anemic fetuses indicating reduced myoblast proliferation. There was less MYOD and MYF6 mRNA expression in anemic vs. control BF consistent with reduced myoblast differentiation. These results indicate that fetal anemic hypoxemia reduced muscle growth. We speculate that fetal muscle growth may be improved by strategies that increase oxygen availability.

Effects of Encapsulated Methionine on Skeletal Muscle Growth and Development and Subsequent Feedlot Performance and Carcass Characteristics in Beef Steers

Two studies were conducted to evaluate the effect of encapsulated methionine on live performance, carcass characteristics, and skeletal muscle development in feedlot steers. In Experiment 1, 128 crossbred steers (body weight [BW] = 341 ± 36.7 kg) were used in a randomized complete block design and supplemented with 0, 4, 8, or 12 g/(head day [d]) of ruminally protected methionine (0MET, 4MET, 8MET, and 12MET, respectively) for 111 d or 139 d. In Exp. 2, 20 steers (BW = 457 ± 58 kg) were stratified by BW and randomly assigned to either the 0MET or 8MET treatment; longissimus muscle (LM) biopsies were collected on d 0, 14, 28, 42, and 56, and analyzed for mRNA and protein expression. Additionally, immunohistochemical analysis was performed to measure fiber type area and distribution as well as the density of muscle nuclei and satellite cells (Myf5, Pax7, and Myf5/Pax7). In Experiment 1, no significant differences were observed for live performance (p ≥ 0.09). There was, however, a linear relationship between LM area and methionine supplementation (p = 0.04), with a 9% increase in the area when steers were supplemented with 12MET compared to 0MET. In Exp. 2, There were no treatment × day interactions (p ≥ 0.10) for expression of mRNA or protein abundance. Although mRNA expression and protein abundance of all genes were influenced by day (p ≤ 0.04), methionine supplementation did not have a significant effect (p ≥ 0.08). There was a significant treatment × day interaction for distribution of MHC-I fibers (p = 0.03), where 8MET supplemented cattle had a greater proportion of MHC-I fibers after 56 d of supplementation than did 0MET steers. Cross-sectional area was increased over time regardless of fiber type (p < 0.01) but was unaffected by treatment (p ≥ 0.36). While nuclei density was not impacted by treatment (p = 0.55), the density of myonuclei increased nearly 55% in 8MET supplemented cattle (p = 0.05). The density of Myf5 positive satellite cells tended to decrease with methionine supplementation (p = 0.10), while the density of Pax7 expressing cells tended to increase (p = 0.09). These results indicate that encapsulated methionine supplementation may influence markers of skeletal muscle growth, and potential improvements in the LM area may exist.

Genome-Wide Identification, Characterization and Expression Profiling of myosin Family Genes in Sebastes schlegelii

Myosins are important eukaryotic motor proteins that bind actin and utilize the energy of ATP hydrolysis to perform a broad range of functions such as muscle contraction, cell migration, cytokinesis, and intracellular trafficking. However, the characterization and function of myosin is poorly studied in teleost fish. In this study, we identified 60 myosin family genes in a marine teleost, black rockfish (Sebastes schlegelii), and further characterized their expression patterns. myosin showed divergent expression patterns in adult tissues, indicating they are involved in different types and compositions of muscle fibers. Among 12 subfamilies, S. schlegelii myo2 subfamily was significantly expanded, which was driven by tandem duplication events. The up-regulation of five representative genes of myo2 in the skeletal muscle during fast-growth stages of juvenile and adult S. schlegelii revealed their active role in skeletal muscle fiber synthesis. Moreover, the expression regulation of myosin during the process of myoblast differentiation in vitro suggested that they contribute to skeletal muscle growth by involvement of both myoblast proliferation and differentiation. Taken together, our work characterized myosin genes systemically and demonstrated their diverse functions in a marine teleost species. This lays foundation for the further studies of muscle growth regulation and molecular mechanisms of indeterminate skeletal muscle growth of large teleost fishes.

Comprehensive analysis of differentially expressed circRNAs and ceRNA regulatory network in porcine skeletal muscle

Background Circular RNA (circRNA), a novel class of non-coding RNA, has a closed-loop structure with important functions in skeletal muscle growth. The purpose of this study was to investigate the role of differentially expressed circRNAs (DEcircRNAs), as well as the DEcircRNA-miRNA-mRNA regulatory network, at different stages of porcine skeletal muscle development. Here, we present a panoramic view of circRNA expression in porcine skeletal muscle from Large White and Mashen pigs at 1, 90, and 180 days of age. Results We identified a total of 5819 circRNAs. DEcircRNA analysis at different stages showed 327 DEcircRNAs present in both breeds. DEcircRNA host genes were concentrated predominately in TGF-β, MAPK, FoxO, and other signaling pathways related to skeletal muscle growth and fat deposition. Further prediction showed that 128 DEcircRNAs could bind to 253 miRNAs, while miRNAs could target 945 mRNAs. The constructed ceRNA network plays a vital role in skeletal muscle growth and development, and fat deposition. Circ_0015885/miR-23b/SESN3 in the ceRNA network attracted our attention. miR-23b and SESN3 were found to participate in skeletal muscle growth regulation, also playing an important role in fat deposition. Using convergent and divergent primer amplification, RNase R digestion, and qRT-PCR, circ_0015885, an exonic circRNA derived from Homer Scaffold Protein 1 (HOMER1), was confirmed to be differentially expressed during skeletal muscle growth. In summary, circ_0015885 may further regulate SESN3 expression by interacting with miR-23b to function in skeletal muscle. Conclusions This study not only enriched the circRNA library in pigs, but also laid a solid foundation for the screening of key circRNAs during skeletal muscle growth and intramural fat deposition. In addition, circ_0015885/miR-23b/SESN3, a new network regulating skeletal muscle growth and fat deposition, was identified as important for increasing the growth rate of pigs and improving meat quality.

Phytochemicals in Chinese chive (Allium tuberosum) Induce the Skeletal Muscle Cell Proliferation via PI3K/Akt/mTOR and Smad Pathways in C2C12 Cells

Chinese chive (Allium tuberosum) is a medicinal food that is cultivated and consumed mainly in Asian countries. Its various phytochemicals and physiological effects have been reported, but only a few phytochemicals are available for skeletal muscle cell proliferation. Herein, we isolated a new compound, kaempferol-3-O-(6″-feruloyl)-sophoroside (1), along with one known flavonoid glycoside (2) and six amino acid (3–8) compounds from the water-soluble fraction of the shoot of the Chinese chive. The isolated compounds were identified using extensive spectroscopic methods, including 1D and 2D NMR, and evaluated for their proliferation activity on skeletal muscle cells. Among the tested compounds, newly isolated flavonoid (1) and 5-aminouridine (7) up-regulated PI3K/Akt/mTOR pathways, which implies a positive effect on skeletal muscle growth and differentiation. In particular, compound 1 down-regulated the Smad pathways, which are negative regulators of skeletal muscle growth. Collectively, we suggest that major constituents of Chinese chive, flavonoids and amino acids, might be used in dietary supplements that aid skeletal muscle growth.