Different Types Of Muscle Fibers Research Articles

Identification of different myofiber types in pigs muscles and construction of regulatory networks

BackgroundSkeletal muscle is composed of muscle fibers with different physiological characteristics, which plays an important role in regulating skeletal muscle metabolism, movement and body homeostasis. The type of skeletal muscle fiber directly affects meat quality. However, the transcriptome and gene interactions between different types of muscle fibers are not well understood.ResultsIn this paper, we selected 180-days-old Large White pigs and found that longissimus dorsi (LD) muscle was dominated by fast-fermenting myofibrils and soleus (SOL) muscle was dominated by slow-oxidizing myofibrils by frozen sections and related mRNA and protein assays. Here, we selected LD muscle and SOL muscle for transcriptomic sequencing, and identified 312 differentially expressed mRNA (DEmRs), 30 differentially expressed miRNA (DEmiRs), 183 differentially expressed lncRNA (DElRs), and 3417 differentially expressed circRNA (DEcRs). The ceRNA network included ssc-miR-378, ssc-miR-378b-3p, ssc-miR-24-3p, XR_308817, XR_308823, SMIM8, MAVS and FOS as multiple core nodes that play important roles in muscle development. Moreover, we found that different members of the miR-10 family expressed differently in oxidized and glycolytic muscle fibers, among which miR-10a-5p was highly expressed in glycolytic muscle fibers (LD) and could target MYBPH gene mRNA. Therefore, we speculate that miR-10a-5p may be involved in the transformation of muscle fiber types by targeting the MYHBP gene. In addition, PPI analysis of differentially expressed mRNA genes showed that ACTC1, ACTG2 and ACTN2 gene had the highest node degree, suggesting that this gene may play a key role in the regulatory network of muscle fiber type determination.ConclusionsWe can conclude that these genes play a key role in regulating muscle fiber type transformation. Our study provides transcriptomic profiles and ceRNA interaction networks for different muscle fiber types in pigs, providing reference for the transformation of pig muscle fiber types and the improvement of meat quality.

Exercise couples mitochondrial function with skeletal muscle fiber type via ROS-mediated epigenetic modification

Skeletal muscle is a heterogeneous tissue composed of different types of muscle fibers, demonstrating substantial plasticity. Physiological or pathological stimuli can induce transitions in muscle fiber types. However, the precise regulatory mechanisms behind these transitions remains unclear. This paper reviews the classification and characteristics of muscle fibers, along with the classical mechanisms of muscle fiber type transitions. Additionally, the role of exercise-induced muscle fiber type transitions in disease intervention is reviewed. Epigenetic pathways mediate cellular adaptations and thus represent potential targets for regulating muscle fiber type transitions. This paper focuses on the mechanisms by which epigenetic modifications couple mitochondrial function and contraction characteristics. Reactive Oxygen Species (ROS) are critical signaling regulators for the health-promoting effects of exercise. Finally, we discuss the role of exercise-induced ROS in regulating epigenetic modifications and the transition of muscle fiber types.

Dietary oleic acid intake increases the proportion of type 1 and 2X muscle fibers in mice

Skeletal muscle is one of the largest metabolic tissues in mammals and is composed of four different types of muscle fibers (types 1, 2A, 2X, and 2B); however, type 2B is absent in humans. Given that slow-twitch fibers are superior to fast-twitch fibers in terms of oxidative metabolism and are rich in mitochondria, shift of muscle fiber types in direction towards slower fiber types improves metabolic disorders and endurance capacity. We previously had reported that oleic acid supplementation increases type 1 fiber formation in C2C12 myotubes; however, its function still remains unclear. This study aimed to determine the effect of oleic acid on the muscle fiber types and endurance capacity. An in vivo mouse model was used, and mice were fed a 10% oleic acid diet for 4 weeks. Two different skeletal muscles, slow soleus muscle with the predominance of slow-twitch fibers and fast extensor digitorum longus (EDL) muscle with the predominance of fast-twitch fibers, were used. We found that dietary oleic acid intake improved running endurance and altered fiber type composition of muscles, the proportion of type 1 and 2X fibers increased in the soleus muscle and type 2X increased in the EDL muscle. The fiber type shift in the EDL muscle was accompanied by an increased muscle TAG content. In addition, blood triacylglycerol (TAG) and non-esterified fatty acid levels decreased during exercise. These changes suggested that lipid utilization as an energy substrate was enhanced by oleic acid. Increased proliferator-activated receptor γ coactivator-1β protein levels were observed in the EDL muscle, which potentially enhanced the fiber type transitions towards type 2X and muscle TAG content. In conclusion, dietary oleic acid intake improved running endurance with the changes of muscle fiber type shares in mice. This study elucidated a novel functionality of oleic acid in skeletal muscle fiber types. Further studies are required to elucidate the underlying mechanisms. Our findings have the potential to contribute to the field of health and sports science through nutritional approaches, such as the development of supplements aimed at improving muscle function.

Decoding the transcriptome of muscular dystrophy due to Ptrf deficiency using single-nucleus RNA sequencing.

Lacking PTRF (polymerase I and transcript release factor), an essential caveolae component, causes a secondary deficiency of caveolins resulting in muscular dystrophy. The transcriptome responses of different types of muscle fibers and mononuclear cells in skeletal muscle to muscular dystrophy caused by Ptrf deletion have not been explored. Here, we created muscular dystrophy mice by Ptrf knockout and applied single-nucleus RNA sequencing (snRNA-seq) to unveil the transcriptional changes of the skeletal muscle at single-nucleus resolution. 11 613 muscle nuclei (WT, 5838; Ptrf KO, 5775) were classified into 12 clusters corresponding to 11 nuclear types. Trajectory analysis revealed the potential transition between type IIb_1 and IIb_2 myonuclei upon muscular dystrophy. Functional enrichment analysis indicated that apoptotic signaling and enzyme-linked receptor protein signaling pathway were significantly enriched in type IIb_1 and IIb_2 myonuclei of Ptrf KO, respectively. The muscle structure development and the PI3K-AKT signaling pathway were significantly enriched in type IIa and IIx myonuclei of Ptrf KO. Meanwhile, metabolic pathway analysis showed a decrease in overall metabolic pathway activity of myonuclei subtypes upon muscular dystrophy, with the most decrease in type IIb_1 myonuclei. Gene regulatory network analysis found that the activity of Mef2c, Mef2d, Myf5, and Pax3 regulons was enhanced in type II myonuclei of Ptrf KO, especially in type IIb_2 myonuclei. In addition, we investigated the transcriptome changes in adipocytes and found that muscular dystrophy enhanced the lipid metabolic capacity of adipocytes. Our findings provide a valuable resource for exploring the molecular mechanism of muscular dystrophy due to Ptrf deficiency.

Sympathetic innervation and endogenous catecholamines in neuromuscular preparations of muscles of different functional profiles

Influence of the sympathetic nervous system on the work of skeletal muscles contractile apparatus is now beyond doubt. However, until recently there was no evidence that the endings of sympathetic nerves can be located in close proximity to the neuromuscular synapses, and there is also no reliable data on how much endogenous adrenaline and noradrenaline can be contained near the synaptic contact in skeletal muscles. In this research, using fluorescent analysis, immunohistochemical and enzyme immunoassays the isolated neuromuscular preparations of three skeletal muscles of different functional profiles and containing different types of muscle fibers were examined. Close contact between the sympathetic and motor cholinergic nerve endings and the presence of tyrosine hydroxylase in this area were demonstrated. Concentrations of endogenous adrenaline and noradrenaline in the solution perfusing the neuromuscular preparation were determined under different modes of its functioning. The effects of α and β adrenoreceptor blockers on the processes of acetylcholine quantal secretion from the motor nerve endings were compared. The data obtained provide evidence for the presence of endogenous catecholamines in the neuromuscular junction region and their role in the modulation of the synaptic function.

Sympathetic Innervation and Endogenous Catecholamines in Neuromuscular Preparations of Muscles with Different Functional Profiles

Influence of the sympathetic nervous system on the work of skeletal muscles contractile apparatus is now beyond doubt. However, until recently there was no evidence that the endings of sympathetic nerves can be located in close proximity to the neuromuscular synapses, and there is also no reliable data on how much endogenous adrenaline and noradrenaline can be contained near the synaptic contact in skeletal muscles. In this research, using fluorescent analysis, immunohistochemical and enzyme immunoassays the isolated neuromuscular preparations of three skeletal muscles of different functional profiles and containing different types of muscle fibers were examined. Close contact between the sympathetic and motor cholinergic nerve endings and the presence of tyrosine hydroxylase in this area were demonstrated. Concentrations of endogenous adrenaline and noradrenaline in the solution perfusing the neuromuscular preparation were determined under different modes of its functioning. The effects of α and β adrenoreceptor blockers on the processes of acetylcholine quantal secretion from the motor nerve endings were compared. The data obtained provide evidence for the presence of endogenous catecholamines in the neuromuscular junction region and their role in modulation of the synaptic function.

The Effects of Branched-Chain Amino Acids on the Akt/mTOR Pathway and Nebulin Protein in Joint Fixation-Induced Muscle Atrophy.

It is well known that branched-chain amino acids (BCAAs) promote protein synthesis in skeletal muscle and can cause muscle hypertrophy. However, it has also been reported that they may inhibit muscle atrophy induced by load-bearing and age-related changes. In this study, we investigated the effects of BCAA intake during joint fixation on the levels of protein kinase B (Akt), mammalian target of rapamycin (mTOR), and nebulin in a rat model of joint fixation. Akt and mTOR are signal factors of protein synthesis, whereas nebulin is a structural protein in the muscle. The effects of BCAAs on muscle atrophy were also investigated. The phosphorylation rate of mTOR was higher than that of Akt and increased with BCAA intake in the rat hind limb muscles (soleus) when the ankle joint was fixed. The relative level of nebulin and the phosphorylation rate of Neural Wiskott-Aldrich syndrome protein (N-WASP) also increased as a result of BCAA intake during fixation. This is important because nebulin and N-WASP are involved in the formation of the structure of sarcomere thin filaments. Furthermore, when the cross-sectional areas (CSAs) of different types of muscle fibers were measured during histological evaluation of muscle atrophy, it was found that the inhibitory effect of BCAA on muscle atrophy was higher in Type 1 fibers. Additionally, a positive correlation was found between nebulin level and the CSAs of the muscle fibers. It was found that there is a close relationship between the content of structural proteins and muscle atrophy.

MiR-208b Regulates the Conversion of Skeletal Muscle Fiber Types by Inhibiting Mettl8 Expression

Skeletal muscle, the main source of animal meat products, contains muscle fiber as a key unit. It is well known that transformation takes place between different types of muscle fibers, however, the conversion mechanism is not clear. In a previous study, our lab has demonstrated that there is a decrease in type I muscle fibers and an increase in type IIB muscle fibers in skeletal muscle of myostatin gene-edited Meishan pigs. Very interestingly, we observed the down regulation of miR-208b expression and an increase in expression the predicted target gene Mettl8 (Methyltransferase like 8) in skeletal muscle of MSTN gene-edited Meishan pigs. These results reveal that there is a potential connection between the conversion of skeletal muscle fiber types and miR-208b and Mettl8 expression. In this study, we first explored the expression patterns of miR-208b and Mettl8 in skeletal muscle in Meishan pigs; and then C2C12 cells were used to simulate the development and maturation of muscle fibers. Our results indicated that Myh4 expression level decreased and Myh7 expression level increased following overexpression of miR-208b in C2C12 cells. We therefore speculate that miR-208b can promote the conversion of fast-twitch fibers to slow-twitch fibers. The targeting relationship between Mettl8 and miR-208b was confirmed by results obtained using dual luciferase assay, RT-qPCR, and WB analysis. Following the transfection of Mettl8 siRNA into C2C12 cells, we observed that Mettl8 expression decreased significantly while Myh7 expression increased and Myh4 expression decreased, indicating that Mettl8 promotes the conversion of slow muscle fibers to fast muscle fibers. Additionally, changes in skeletal muscle fiber types are observed in those mice where miR-208b and Mettl8 genes are knocked out. The miR-208b knockout inhibits the formation of slow muscle fibers, and the Mettl8 knockout inhibits the formation of fast muscle fibers. In conclusion, our research results show that miR-208b regulates the conversion of different muscle fiber types by inhibiting Mettl8 expression.

An index to measure the activity attitude of broilers in extensive system

In organic poultry production it is important to rear animals with a dynamic attitude to take advantage of outdoor areas. Farmers are reluctant to use such strains due to their lower productivity and older slaughtering age. However, fast growing lines grown in organic system often suffer poor health and welfare conditions. The kinetic metabolism of chickens is correlated with different types of muscle fiber: type I (in red muscles or oxidative) for prolonged and moderate movement and type II (in white muscles or glycogenic) for fast movements. Red muscle metabolism produces energy mainly by β-oxidation of Highly Unsaturated n-3 Fatty Acids (HUFA). Accordingly, kinetic activity causes higher consume of HUFA in red muscles than in white muscles, so the ratio between n-3 HUFA and their precursor C18:3 n-3 (ALA) is likely to be smaller in red than in white muscles. However, these ratios are highly affected by the environment. To reduce the effect of environmental variables, we propose an “Activity index” as the difference between n-3 HUFA/ALA in white and red muscle within the same bird. This index, measured after slaughtering, should represent the activity performed by the chicken during its life. Given that birds in good health had the possibility of moving, the “Activity index” would measure the activity actually performed by the animals. Should birds of a given strain show a higher activity level, this would be an indication of the suitability of that strain to outdoor systems. This work verified the application of this “Activity index” on 90 birds from 6 genetic strains with known kinetic behavior reared in an experimental farm. The “Activity index” was also tested on chicken strains collected form commercial organic farms. The results confirmed that strains recognized for higher kinetic attitude actually walked more and their behavior was clearly detected by the “Activity index” estimated from their muscles.

Histopathological changes and oxidative damage in type I and type II muscle fibers in rats undergoing paradoxical sleep deprivation

Backgroundprevious studies have shown that muscle atrophy is observed after sleep deprivation (SD) protocols; however, the mechanisms responsible are not fully understood. Muscle trophism can be modulated by several factors, including energy balance (positive or negative), nutritional status, oxidative stress, the level of physical activity, and disuse. The metabolic differences that exist in different types of muscle fiber may also be the result of different adaptive responses. To better understand these mechanisms, we evaluated markers of oxidative damage and histopathological changes in different types of muscle fibers in sleep-deprived rats. MethodsTwenty male Wistar EPM-1 rats were randomly allocated in two groups: a control group (CTL group; n = 10) and a sleep deprived group (SD group; n = 10). The SD group was submitted to continuous paradoxical SD for 96 h; the soleus (type I fibers) and plantar (type II fiber) muscles were analyzed for histopathological changes, trophism, lysosomal activity, and oxidative damage. Oxidative damage was assessed by lipid peroxidation and nuclear labeling of 8-OHdG. ResultsThe data demonstrated that SD increased the nuclear labeling of 8-OHdG and induced histopathological changes in both muscles, being more evident in the soleus muscle. In the type I fibers there was signs of tissue degeneration, inflammatory infiltrate and tissue edema. Muscle atrophy was observed in both muscles. The concentration of malondialdehyde, and cathepsin L activity only increased in type I fibers after SD. ConclusionThese data indicate that the histopathological changes observed after 96 h of SD in the skeletal muscle occur by different processes, according to the type of muscle fiber, with muscles predominantly composed of type I fibers undergoing greater oxidative damage and catabolic activity, as evidenced by a larger increase in 8-OHdG labeling, lipid peroxidation, and lysosomal activity.

Regular aerobic exercise increased VEGF levels in both soleus and gastrocnemius muscles correlated with hippocampal learning and VEGF levels.

Physical exercise improves learning and memory abilities by increasing the levels of several growth factors in the hippocampus. One growth factor, vascular endothelial growth factor (VEGF), is primarily produced in the muscles and not only increases in the periphery during exercise but can also cross the blood-brain barrier. The aim of this study is to investigate the effects of regular aerobic chronic exercise on different types of muscle fibers and the relationships between learning/memory and muscle induced-VEGF. Following a one-week adaptation period, male rats underwent treadmill training at a speed of 8 m/min for 30 min daily, 3 days a week for 6 weeks. Memory functions were evaluated using the Morris water maze. VEGF, superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) levels were measured in type 1 and type 2 muscle fibers and VEGF levels were also measured in the hippocampus. Exercise positively affected both learning and memory and also increased VEGF levels in both muscle fiber types. Muscle VEGF levels positively correlate with hippocampal learning and hippocampal VEGF levels. Exercise reduced both SOD and MDA levels in type 1 and type 2 muscle fibers, whereas GPx levels decreased only in type 2 muscle fibers. Our findings suggest that regular aerobic exercise elevates VEGF levels and diminishes oxidative stress in both fiber types. Exercise-induced VEGF levels in both type 1 and 2 muscle fibers appear to be associated with the positive effect of exercise on learning and memory function and is accompanied by an increase in VEGF levels in the hippocampus. Further research is needed to elucidate the exact mechanism by which fiber type-specific VEGF mediates hippocampal neurogenesis and angiogenesis. Physical exercise improves learning and memory abilities by increasing the levels of several growth factors in the hippocampus. One growth factor, vascular endothelial growth factor (VEGF), is primarily produced in the muscles and not only increases in the periphery during exercise but can also cross the blood-brain barrier. The aim of this study is to investigate the effects of regular aerobic chronic exercise on different types of muscle fibers and the relationships between learning/memory and muscle induced-VEGF. Following a one-week adaptation period, male rats underwent treadmill training at a speed of 8 m/min for 30 min daily, 3 days a week for 6 weeks. Memory functions were evaluated using the Morris water maze. VEGF, superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA) levels were measured in type 1 and type 2 muscle fibers and VEGF levels were also measured in the hippocampus. Exercise positively affected both learning and memory and also increased VEGF levels in both muscle fiber types. Muscle VEGF levels positively correlate with hippocampal learning and hippocampal VEGF levels. Exercise reduced both SOD and MDA levels in type 1 and type 2 muscle fibers, whereas GPx levels decreased only in type 2 muscle fibers. Our findings suggest that regular aerobic exercise elevates VEGF levels and diminishes oxidative stress in both fiber types. Exercise-induced VEGF levels in both type 1 and 2 muscle fibers appear to be associated with the positive effect of exercise on learning and memory function and is accompanied by an increase in VEGF levels in the hippocampus. Further research is needed to elucidate the exact mechanism by which fiber type-specific VEGF mediates hippocampal neurogenesis and angiogenesis.

Comparing the mRNA Expression Profile of Psoas Major and Longissimus Dorsi Muscles in Pig

To explore the differentially expressed mRNAs between oxidative and glycolytic muscles, Qianbei black pigs were slaughtered and longissimus dorsi muscle (LDM) and psoas major muscle (PMM) were selected and sequenced using Illumina Hiseq TM 4000. Bioinformatics analysis and differentially expressed genes were analyzed by GO and KEGG. qRT-PCR was used to validate the RNA-seq result. As a result, 69 differentially expressed genes were identified, with 46 down regulated genes and 23 up regulated genes in LDM versus PMM, which were categorized into 44 functional groups under three GO classifications. KEGG pathway analysis revealed 20 pathways were enriched. qRT-PCR shows the expression trends of ND6, MYH7, TBX1, FOS and SLC7A5 are consist with the RNA-seq result. We speculated these five genes may involve in differentiation of muscle cells, metabolism of carbohydrate and lipid, deposits of intramuscular fat and transformation of different types of muscle fibers.

The long noncoding RNA MyHC IIA/X-AS contributes to skeletal muscle myogenesis and maintains the fast fiber phenotype

Mammalian skeletal muscles comprise different types of muscle fibers, and this muscle fiber heterogeneity is generally characterized by the expression of myosin heavy chain (MyHC) isoforms. A switch in MyHC expression leads to muscle fiber-type transition under various physiological and pathological conditions, but the underlying regulator coordinating the switch of MyHC expression remains largely unknown. Experiments reported in this study revealed the presence of a skeletal muscle-specific antisense transcript generated from the intergenic region between porcine MyHC IIa and IIx and is referred to here as MyHC IIA/X-AS. We found that MyHC IIA/X-AS is identified as a long noncoding RNA (lncRNA) that is strictly expressed in skeletal muscles and is predominantly distributed in the cytoplasm. Genetic analysis disclosed that MyHC IIA/X-AS stimulates cell cycle exit of skeletal satellite cells and their fusion into myotubes. Moreover, we observed that MyHC IIA/X-AS is more enriched in fast-twitch muscle and represses slow-type gene expression and thereby maintains the fast phenotype. Furthermore, we found that MyHC IIA/X-AS acts as a competing endogenous RNA that sponges microRNA-130b (miR-130b) and thereby maintains MyHC IIx expression and the fast fiber type. We also noted that miR-130b was proved to down-regulate MyHC IIx by directly targeting its 3'-UTR. Together, the results of our study uncovered a novel pathway, which revealed that lncRNA derived from the skeletal MyHC cluster could modulate local MyHC expression in trans, highlighting the role of lncRNAs in muscle fiber-type switching.

Maintenance of spontaneous breathing at an intensity of 60%-80% may effectively prevent mechanical ventilation-induced diaphragmatic dysfunction.

Controlled mechanical ventilation (CMV) can cause diaphragmatic motionlessness to induce diaphragmatic dysfunction. Partial maintenance of spontaneous breathing (SB) can reduce ventilation-induced diaphragmatic dysfunction (VIDD). However, to what extent SB is maintained in CMV can attenuate or even prevent VIDD has been rarely reported. The current study aimed to investigate the relationship between SB intensity and VIDD and to identify what intensity of SB maintained in CMV can effectively avoid VIDD. Adult rats were randomly divided according to different SB intensities: SB (0% pressure controlled ventilation (PCV)), high-intensity SB (20% PCV), medium-intensity SB (40% PCV), medium-low intensity SB (60% PCV), low-intensity SB (80% PCV), and PCV (100% PCV). The animals underwent 24-h controlled mechanical ventilation (CMV). The transdiaphragmatic pressure (Pdi), the maximal Pdi (Pdi max) when phrenic nerves were stimulated, Pdi/Pdi max, and the diaphragmatic tonus under different frequencies of electric stimulations were determined. Calpain and caspase-3 were detected using ELISA and the cross-section areas (CSAs) of different types of muscle fibers were measured. The Pdi showed a significant decrease from 20% PCV and the Pdi max showed a significant decrease from 40% PCV (P<0.05). In vivo and vitro diaphragmatic tonus exhibited a significant decrease from 40% PCV and 20% PCV, respectively (P<0.05). From 20% PCV, the CSAs of types I, IIa, and IIb/x muscle fibers showed significant differences, which reached the lowest levels at 100% PCV. SB intensity is negatively associated with the development of VIDD. Maintenance of SB at an intensity of 60%-80% may effectively prevent the occurrence of VIDD.

Potential Mechanisms for Marbling Content Differences in M. Longissimus Dorsi From Wagyu And Brahman Cattle

ObjectivesIntramuscular fat (marbling) affects consumer perceptions of meat quality. Recent studies indicated that intramuscular adipocytes are derived from fibroadipogenic progenitors (FAPs), a type of bipotent mesenchymal progenitor cells in extracellular matrix of muscle fiber. Platelet-derived growth factor receptor α (PDGFRa) is a marker in identifying FAPs. The amount of PDGFRa positive cells refers to the abundance of FAPs, a key factor determining the intramuscular adipogenic efficiency of the animal. In addition, it has been shown that both satellite cells and skeletal muscle fibers regulate the activity of FAPs. In this study, we aimed to identify some underlying mechanisms for the differences in intramuscular fat accumulation between Wagyu, a high marbling breed, and Brahman, a low marbling breed, through a comparison study.Materials and MethodsFive cattle of each breed approaching mature body composition (Wagyu born January to May 2017; Brahman two 2-yr-old and three 3-yr-old, born April 2017 and 2015) were selected for this study. Biopsy samples of M. Longissimus muscle (LM) were taken after local anesthesia between the 12th and 13th ribs. Fresh samples were frozen in isopentane chilled by liquid nitrogen immediately after trimming fat and connective tissue. Other samples were fixed for 4 h in ice-cold 4% paraformaldehyde, soaked overnight in 30% sucrose solution, and then processed in Optimal Cutting Temperature embedding medium. Frozen samples were stored at –80°C before cryosectioning (5 mm) at –25°C for immunochemical staining. Unfixed muscle tissue sections were only used for muscle fiber determinations by rinsing with 1X Tris Buffer Saline (TBS) 3 times, 5 min each, before blocking in 10% goat serum and 1% bovine serum albumin, followed by overnight incubation with primary antibodies at 4°C. Subsequent secondary antibody staining at room temperature for 1 h. TBS/T (0.3% Triton) was used for membrane permeabilization to identify satellite cells, FAPs, and basement membrane. Mounting media containing 4,6-diamidino-2-phenylindole (DAPI) was used for nucleus staining. Primary antibodies against PDGFRα, paired box 7 (PAX7), laminin, and different isoforms of skeletal muscle myosins were used to identify FAPs, satellite cells, basement membrane, and different types of muscle fiber, respectively. All the sections were visualized under a Nikon inflorescence microscope, and images were analyzed by Image J software to identify different muscle fiber types and positive signals of PDGFRα and PAX7. Minimum diameter of each muscle fiber type was the average of 30 randomly selected fibers per section. Data were analyzed with R-studio using ‘t.test ()’ function with the critical value being equal to 0.05.ResultsWagyu muscle demonstrated a greater (P = 0.01) number of FAPs compared with Brahman muscle. A trend toward a higher (P = 0.06) abundance of satellite cells in Wagyu muscle than in Brahman muscles was also identified. No differences were found in muscle fiber diameter or muscle fiber type composition between these two breeds of cattle.ConclusionThe greater number of FAP cells observed in the LM of Wagyu cattle than in Brahman cattle suggests that the higher marbling content of Wagyu meat is at least partially attributed to the more abundant adipogenic progenitor cells, which increases the capacity and efficiency of intramuscular adipogenesis in Wagyu cattle.

Effect of production factors on muscle fiber type and dimensions in the m. semimembranosus of crossbred steer carcasses

The muscle fibers that have been examined in the study were affected by three different controlled factors: steroids, ractopamine and residual feed intake (RFI). By examining the effects of the controlled factors on cattle’s muscle fibers, it can be determined if they affect different meat properties, such as meat toughness, collagen solubility and muscle fiber quality. The research had been done specifically with m. semimembranosus (SM) of crossbred steers. Although some may be concerned with the health effects of steroids and other materials, no negative effects to the health of the cattle were observed after the use of steroids. This is because the hormones being introduced into the cattle’s body already exist in the animal. In addition, the same concept applies to humans who consume the meat, preventing harm the people who consume it. For this study, 48 crossbred angus steers were used, 12 for each of the different treatment groups. The control group consisted of no steroids and no ractopamine. The second group was not treated with steroid but with ractopamine. The third group was treated with steroids but no ractopamine. Finally, the fourth group was treated with both, the steroids and the ractopamine. For each SM muscle, 1-inch thick steaks were cut and from those steaks, 1cm3 cubes were cut. These cubes were frozen in dry ice acetone until they are ready to be sectioned. Cubes are placed in the cryostat and sliced into serial sections of 10µm. These serial sections are then mounted onto dry slide glass and stored in a freezer at -80ºC until they are to be stained. The staining process helps to identify the different types of muscle fibers in the samples. From the muscle fiber types, the average sizes of each muscle fiber is calculated to identify inconsistencies among the different treatment groups. Conclusions will be drawn based on the inconsistencies found (if any).

The Correlation between Osteoporosis and Blood Circulation Function Based on Magnetic Resonance Imaging.

In order to investigate the relationship between changes in blood circulation and bone mineral density (BMD) loss, the characteristic parameters reflecting the function of tissue oxygen metabolism are obtained by means of blood oxygen level-dependent magnetic resonance imaging (BOLD-MRI), image processing and semi-quantitative analysis. The correlation and variance analysis of the characteristic parameters of different BMD groups are carried out, and the physiological parameters of bone marrow blood perfusion are obtained by dynamic enhanced MRI (DCE-MRI). Multivariate logistic regression analysis is carried out with the physiological parameters of blood oxygen metabolism function and bone marrow blood perfusion as independent variables and BMD as dependent variables. It is found that there are significant differences in oxygen metabolism between individual muscles in different BMD groups and between skeletal muscles of different types of muscle fibers. Age, total volume of bone marrow and oxygen metabolism ability of tibial anterior muscle have significant independent effects on osteoporosis. It shows that the changes of blood circulation in bone marrow and surrounding muscle tissue are indeed one of the causes of osteoporosis.

Immunohistochemical Identification of Muscle Fiber Types in Mice Tibialis Anterior Sections.

Mammalian skeletal muscle is a metabolically active tissue that is made up of different types of muscle fibers. These myofibers are made up of important contractile proteins that provide force during contraction of the muscle like actin and myosin. Murine myofibers have been classified into 4 types: Type I, Type IIa, Type IIb and Type IIX. Each muscle fiber has been identified with specific type of MyHC expressed, which in turn gives differential contractility to the muscle. There have been well-known methodologies to identify different myofibers: histochemical myosin ATPase staining which uses the differential ATPase activity between slow and fast fibers, quantification of metabolic enzymes like malate dehydrogenase and lactate dehydrogenase on specific fragments of muscle fibers. The drawback of these techniques is that they cannot differentiate the subtypes of myofibers, for example, Type IIa and Type IIb. They should be used in conjunction with other known histochemical staining techniques. Here, we devise a direct and robust immunohistochemical staining methodology that utilizes the differential expression of MyHC isoforms in different myofibers types, thus efficiently distinguishing the heterogeneity of the muscle fibers. We use antibodies that specifically recognize Type I, Type IIa and Type IIb fibers on serially cut frozen mouse tibialis anterior sections that can be quantified by ImageJ software.

Muscle spindles of the rat sternomastoid muscle.

The sternomastoid (SM) muscle in rodents presents a peculiar distribution of fiber types with a steep gradient from the ventral, superficial, white portion to the dorsal, deep, red region, where muscle spindles are restricted. Cross section of the medial longitudinal third of the rat SM contains around 10,000 muscle fibers with a mean diameter of 51.28±12.62 (μm +/- SD). Transverse sections stained by Succinate Dehydrogenase (SDH) reaction clearly presents two distinct regions: the dorsal deep red portion encompassing a 40% cross section area contains a high percentage of packed SDH-positive muscle fibers, and the ventral superficial region which contains mainly SDH-negative muscle fibers. Indeed, the ventral superficial region of the rat SM muscle contains mainly fast 2B muscle fibers. These acidic ATPase pH 4.3-negative and SDH-negative 2B muscle fibers are the largest of the SM muscle, while the acidic ATPase pH 4.3-positive and SDH-positive Type 1 muscle fibers are the smallest. Here we show that in thin transverse cryosections only 2 or 3 muscle spindle are observed in the central part of the dorsal deep red portion of the SM muscle. Azan Mallory stained sections allow at the same time to count the spindles and to evaluate aging fibrosis of the skeletal muscle tissue. Though restricted in the muscle red region, SM spindles are embedded in perimysium, whose changes may influence their reflex activity. Our findings confirm that any comparisons of changes in number and percentage of muscle spindles and muscle fibers of the rat SM muscle will require morphometry of the whole muscle cross-section. Muscle biopsies of SM muscle from large mammals will only provide partial data on the size of the different types of muscle fibers biased by sampling. Nonetheless, histology of muscle tissue continue to provide practical and low-cost quantitative data to follow-up translational studies in rodents and beyond.

PO-089 Effects of 6-days nitrate supplementation on [Ca2+] and CRT in skeletal muscle of exhausted rats

Objective There are many active ingredients in sports nutrition, and nitrate is gradually being valued by sports nutrition and product developers. Supplementation of nitrate is a practical method to increase circulating plasma nitrite, thereby increasing NO bioavailability. However, the existing research has rarely reported the dose effect between nitrate supplementation and changes in exercise efficiency and capacity. The mechanism of action of nitrate is not fully understood yet. The aim of this study was to analyze the effects of different doses of nitrate on the exercise capacity of rats, as well as the detection of [Ca2+] and calreticulin (CRT) expression in the gastrocnemius, soleus and extensor digitorum longus, trying to figure out the effects of different doses of nitrate supplementation on calcium homeostasis in different types of muscle fibers.&#x0D; Methods 40 SD rats (8-week-old) weighing 270-290 grams were randomly divided into control group (C group, 8), exercise control group (EC group, 8), exercise with low-dose supplementation group (ELN group, 8), exercise with medium-dose supplementation group (EMN group, 8) and exercise with high-dose supplementation group (EHN group, 8). Sodium nitrate was used as exogenous nitrate for oral gavage. The intragastric concentration was 0.3 mmol/day/kg body weight in the ELN group, 0.7 mmol/day/kg body weight in the EMN group, and 1.0 mmol/day/kg body weight in the EHN group. The others were orally administered with normal saline. All exercise groups (EC, ELN, EMN and EHN) underwent 3 days adaptive low-intensity treadmill training with slope 0°, speed 16 meter/min, and the time is 5 min, 10 min and 15 min incrementally. 24 hours after the end of the last gavage, a one-time exhaustion treadmill experiment was started. The running platform slope was -16°, the speed was 16 meter/min. Exhaustive experiment participants were not aware of the grouping of rats. Immediately after the end of exhaustive exercise, the rats were weighed and anesthetized with sodium pentobarbital solution. Blood is collected for testing [NO2-]. The gastrocnemius, soleus and extensor digitorum longus were collected for testing NOS activity, tissue [Ca2+] and CRT expression.&#x0D; Results (1) Compared with the EC group, the exhaustion time of the other exercise groups was prolonged. The exhaustion time of the EMN group was very significantly prolonged from that of the EC group (P&lt;0.01). The exhaustion time of the EMN group was significantly prolonged from that of the EC group (P&lt; 0.05). At the same time, the difference between the EMN group and the ELN group was statistically significant (P&lt;0.05). (2) Serum [NO2-] in the supplemented nitrate groups (ELN, EMN and EHN) was higher than that in the EC group, and the difference was statistically significant (P&lt;0.01). The serum [NO2-] in different groups (ELN, EMN and EHN) raised with the increase of nitrate concentration, and the difference between each adjacent concentration group was statistically significant (P&lt;0.05). (3) In the gastrocnemius, soleus and extensor digitorum longus respectively, there were no significant differences in TNOS, iNOS and cNOS activities between the groups (P&gt;0.05). (4) In the gastrocnemius, compared with the EC group, the [Ca2+] (P&lt;0.05) and CRT expression (P&lt;0.05) in the EMN group were significantly decreased. There was no difference between the ELN vs EC group (P&gt;0.05) and the EHN vs EC group (P&gt;0.05). (5) In the soleus, the [Ca2+] (P&lt;0.05) and CRT expression (P&lt;0.05) in the EMN group were significantly decreased. There was no difference between the ELN vs EC group (P&gt;0.05) and the EHN vs EC group (P&gt;0.05). (6) In the extensor digitorum longus, compared with the EC group, the [Ca2+] (P&lt;0.05) and CRT expression (P&lt;0.05) in the EHN group were significantly decreased. There was no difference between ELN and EC group (P&gt;0.05). The expression of CRT in EMN group was significantly decreased (P&lt;0.05), but there is no difference of [Ca2+] between EMN and EC group (P&lt;0.05).&#x0D; Conclusions (1) 6-day sodium nitrate supplementation is a reliable method to increase serum [NO2-] concentration. (2) 6-day sodium nitrate supplementation can prolong the duration of one-time exhaustive exercise in rats, and the dose of 0.7mmol/kg/d is the best. (3) Sodium nitrate supplementation can affect the [Ca2+] and the expression of CRT in skeletal muscle after one-time exhaustive exercise. Different concentrations of sodium nitrate have different effects on different types of muscle fibers.