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Myofiber development during embryonic to neonatal development in duck breeds differing in muscle growth rates
Abstract
Little is known about the muscle developmental patterns during embryonic to neonatal development in ducks. We investigated the developmental patterns in the lateral gastrocnemius muscles of Gaoyou and Jinding ducks differing in their muscle growth rates during the final stages of egg incubation and the first week after hatching. Expression of the MyoD gene was quantified by quantitative real-time PCR (qRT-PCR). The average cross-sectional area and diameter of the fibers increased from embryonic day 21 (E21), peaking at E27, and then declining slightly 7 d after hatching. The density of the fibers decreased initially but increased after hatching in both breeds and sexes. The within-breed variation in muscle fiber-type composition was greater than the average variation between the breeds. Overall, the percentage of type I fibers increased and that of type Ilb fibers decreased consistently. However, the percentage of type Ila fibers was almost constant as development proceeded in both duck breeds. The profiles of MyoD mRNA expression were similar in both breeds, and a significantly positive relationship was observed between the expression of MyoD and the percentage of type Ilb fibers. This study firstly revealed the characteristics of duck muscle development and differences between the two breeds differing in growth rates. Moreover, type Ilb fibers might convert to type I fibers in the lateral gastrocnemius, while MyoD may potentially function in controlling the muscle fiber phenotype during the secondary myogenesis of muscle development.
... The characteristics of muscle fiber are commonly used as essential parameters to evaluate meat quantity and quality during growth and development [33][34][35]. It has been reported that sex had no effect on muscle fiber characteristics [36,37]. Therefore, this study did not separately investigate the effects of gender. ...
... In addition, the myofiber density of breast and leg muscles increased slightly from days 11 to 12 and then showed a downward trend. Similar results were obtained in studies of ducks, where the average density constantly decreased from E21 (embryonic day 21) to E27 (embryonic day 27), indicating that the number of muscle fibers continued to increase from the early to the middle stages of embryonic development, reaching a peak around E21, and then remained relatively fixed [37]. In quail embryos, we found that the number of muscle fibers reached its peak and was basically fixed on embryonic day 12. ...
Simple Summary
The current work revealed the developmental characteristics of skeletal muscle and the expression patterns of related regulatory genes in the embryonic stage of quails. During the embryonic stage, the weight and fiber size of the leg muscle was larger than the breast muscle. The MyoD and Pax7 genes were critical myogenic regulatory factors and were highly expressed in the middle stage of the embryonic period in breast and leg muscles. Overall, these results imply that day 12 of quail embryos may be a crucial point for muscle development.
Abstract
The quail is an important research model, and the demand for quail meat has been increasing in recent years; therefore, it is worthwhile investigating the development of embryonic skeletal muscle and the expression patterns of regulatory genes. In this study, the expression of MyoD and Pax7 in the breast muscle (m. pectoralis major) and leg muscle (m. biceps femoris) of quail embryos on days 10 through 17 were determined using qRT-PCR. Paraffin sections of embryonic muscle were analyzed to characterize changes over time. Results showed that MyoD and Pax7 were expressed in both breast and leg muscles and played a significant role in embryonic muscle development. Compared to breast muscle, leg muscle grew faster and had greater weight and myofiber size. The findings suggested that embryonic day 12 (E12) may be a key point for muscle development. Correlation analysis showed that MyoD expression was significantly negatively correlated with muscle and embryo weight, whereas Pax7 gene expression had no significant correlation with these characteristics. These fundamental results provide a theoretical basis for understanding the characteristics and transition points of skeletal muscle development in quail embryos and an important reference for farmers raising quail from eggs.
... Meat-type and egg-type duck breeds have undergone different genetic selection processes, and thus display significant genetic differences in terms of muscle growth rates and egg production, whilst maintaining consistent developmental processes, e.g. in muscle development pattern. Gaoyou and Jinding ducks comprise important indigenous Chinese meat-type and egg-type duck breeds (average bodyweight approximately 2.48 kg and 1.54 kg at 70 days old for Gaoyou and Jinding ducks, respectively) (Animal Genetic Resources in China-Poultry, 2011) [1]. The different genetic backgrounds in terms of muscle between Gaoyou and Jinding ducks provides a potential model for identifying the mechanisms involved in the muscle-development process. ...
... The PM mass in Gaoyou ducks was significantly higher than in Jinding ducks during the early development period (S1 Fig)[4]. We also analyzed the characteristics (CSA, diameter) of PM in the above two duck breeds and found results that were consistent with our earlier report [1]. This phenomenon was also reported in turkeys by Moore et al. [14], who found that the CSA of myofibers and satellite cell mitotic activity in PM decreased in late-term turkey embryos. ...
Pectoral muscle (PM) comprises an important component of overall meat mass in ducks. However, PM has shown arrested or even reduced growth during late embryonic development, and the molecular mechanisms underlying PM growth during the late embryonic to neonatal period in ducks have not been addressed. In this study, we characterized potential candidate genes and signaling pathways related to PM development using RNA sequencing of PM samples selected at embryonic days (E) 21 and 27 and 5 days post-hatch (dph) in two duck breeds (Gaoyou and Jinding ducks). A total of 393 differentially expressed genes (DEGs) were identified, which showed higher or lower expression levels at E27 compared with E21 and 5 dph, reflecting the pattern of PM growth rates. Among these, 43 DEGs were common to all three time points in both duck breeds. These DEGs may thus be involved in regulating this developmental process. Specifically, KEGG pathway analysis of the 393 DEGs showed that genes involved with different metabolism pathways were highly expressed, while genes involved with cell cycle pathways showed lower expression levels at E27. These DEGs may thus be involved in the mechanisms responsible for the phenomenon of static or decreased breast muscle growth in duck breeds during the late embryonic period. These results increase the available genetic information for ducks and provide valuable resources for analyzing the mechanisms underlying the process of PM development.
... Duck can also be used to control pests in areas where flies are a problem. After 4 weeks ducks can search for their own feed but for better conversion a balanced ration is required [3]. On the same note, the duck price is affordable as compared to other poultry species. ...
A study to assess the effect of replacing Soybean with Pigeon pea on growth performance, meat quality and sensory characteristics of Malawian Indigenous Muscovy ducks was conducted at Bunda Campus of the Lilongwe University of Agriculture and Natural Resources (LUANAR). The study used forty-two (42) ducks which were assigned to two treatments (T1:21 ducks on Soybean meal based-ration, T2:21 ducks on Pigeon pea-based ration). Major feed ingredients were analyzed for nutrient composition before formulating ration. Starter of 20% CP and finisher of 17% CP was fed to ducks in an intensive system. Weekly body weights were measured and used to determine average daily body weight gain which showed better results on pigeon pea fed ducks. Feed conversion ratio were statistically significant with pigeon showing the best value of 2.0 ± 0.00 and Soybean 2.24 ± 0.00. At the 12 th week of age, six ducks from each treatment were selected with reference to mean weight and then slaughtered. Using the right breast muscle, Carcass temperature, and pH were measured at 45minutes, 3hours, 6hours, 12 hours and 24hours post-mortem. Cooking loss, drip loss, and color were determined at 24hours post-mortem. There was no difference between treatments on redness (a*) but the difference was observed on lightness (L*) and yellowness (b*). The left breast muscle was cut from each duck, labelled then chilled at 4 0 C for 24hours followed by cooking at 70 0 C for 15 minutes by boiling. Trained panelists scored the meat samples for sensory characteristics. The results showed that the panelists preferred duck meat from pigeon pea-based ration to Soybean based ration. Therefore, pigeon pea can replace soybean as a protein source since it has a positive effect on Malawian Indigenous Muscovy ducks.
... According to our previous report, myofiber types were identified using myosin ATPase staining (Li et al., 2016). As described by Brooke and Kaiser (1970), type IIB was intensely active and stained dark brown, type IIA was weakly active and stained light brown, and type was inactive and non-stained. ...
MicroRNAs (miRNAs) might play critical roles in skeletal myofiber specification. In a previous study, we found that chicken miR-499-5p is specifically expressed in slow-twitch muscle and that its potential target gene is SOX6. In this study, we performed RNA sequencing to investigate the effects of SOX6 and miR-499-5p on the modulation and regulation of chicken muscle fiber type and its regulatory mechanism. The expression levels of miR-499-5p and SOX6 demonstrated opposing trends in different skeletal muscles and were associated with muscle fiber type composition. Differential expression analysis revealed that miR-499-5p overexpression led to significant changes in the expression of 297 genes in chicken primary myoblasts (CPMs). Myofiber type-related genes, including MYH7B and CSRP3, showed expression patterns similar to those in slow-twitch muscle. According to functional enrichment analysis, differentially expressed genes were mostly associated with muscle development and muscle fiber-related processes. SOX6 was identified as the target gene of miR-499-5p in CPM using target gene mining and luciferase reporter assays. SOX6 knockdown resulted in upregulation of the slow myosin genes and downregulation of fast myosin genes. Furthermore, protein-protein interaction network analysis revealed that MYH7B and RUNX2 may be the direct targets of SOX6. These results indicated that chicken miR-499-5p may promote slow-twitch muscle fiber formation by repressing SOX6 expression. Our study provides a dataset that can be used as a reference for animal meat quality and human muscle disease studies.
... Breast muscle organizational differences among breeds and between sexes begin to occur between ED20 and ED25 in turkeys . While the average cross-sectional area and diameter of the fibers peaks at ED27 in ducks (Li et al., 2016). Thus, embryonic muscle development during the perinatal period has an essential impact on the posthatch accumulation of muscle mass and its infiltration with intramuscular fat (Velleman, 2007). ...
White striping, wooden breast, and spaghetti muscle have become common myopathies in broilers worldwide. Several research reports have indicated that the origin of these lesions is metabolic disorders. These failures in normal metabolism can start very early in life, and suboptimal incubation conditions may trigger some of the key alterations on muscle metabolism. Incubation conditions affect the development of muscle and can be associated with the onset of myopathies. A series of experiments conducted with broilers, turkeys, and ducks are discussed to overview primary information showing the main changes in breast muscle histomorphology, metabolism, and physiology caused by suboptimal incubation conditions. These modifications may be associated with current myopathies. Those effects of incubation on myopathy occurrence and severity have also been confirmed at slaughter age. The impact of egg storage, temperature profiles, oxygen concentrations, and time of hatch have been evaluated. The effects have been observed in diverse species, genetic lines, and both genders. Histological and muscle evaluations have detected that myopathies could be induced by extended hypoxia and high temperatures, and those effects depend on the genetic line. Thus, these modifications in muscle metabolic responses may make hatchlings more susceptible to develop myopathies during grow out due to thermal stress, high-density diets, and fast growth rates.
... Serial tissue sections of 12 mm thickness were prepared and myosin ATPase staining was used to identify the myofiber characteristics. These were carried out according to our previous report [23]. ...
Oxidative and glycolytic myofibers have different structures and metabolic characteristics and their ratios are important in determining poultry meat quality. However, the molecular mechanisms underlying their differences are unclear. In this study, global gene expression profiling was conducted in oxidative skeletal muscle (obtained from the soleus, or SOL) and glycolytic skeletal muscle (obtained from the extensor digitorum longus, or EDL) of Chinese Qingyuan partridge chickens, using the Agilent Chicken Gene Expression Chip. A total of 1224 genes with at least 2-fold differences were identified (P < 0.05), of which 654 were upregulated and 570 were downregulated in SOL. GO, KEGG pathway, and co-expressed gene network analyses suggested that PRKAG3, ATP2A2, and PPARGC1A might play important roles in myofiber composition. The function of PPARGC1A gene was further validated. PPARGC1A mRNA expression levels were higher in SOL than in EDL muscles throughout the early postnatal development stages. In myoblast cells, shRNA knockdown of PPARGC1A significantly inhibited some muscle development and transition-related genes, including PPP3CA, MEF2C, and SM (P < 0.01 or P < 0.05), and significantly upregulated the expression of FWM (P < 0.05). Our study demonstrates strong transcriptome differences between oxidative and glycolytic myofibers, and the results suggest that PPARGC1A is a key gene involved in chicken myofiber composition and transition.
The Jinling White duck represents a newly developed breed characterized by a rapid growth rate and a superior meat quality, offering significant economic value and research potential; however, the genetic basis underlying their body weight traits remains less understood. Here, we performed whole-genome resequencing for 201 diverse Jinling White male ducks and conducted population genomic analyses, suggesting a rich genetic diversity within the Jinling White duck population. Equipped with our genomic resources, we applied genome-wide association analysis for body weight on birth (BWB), body weight on 1 wk (BW1), body weight on 3 wk (BW3), body weight on 5 wk (BW5) and body weight on 7 wk (BW7) using 4 statistical models. Comparative studies indicated that factored spectrally transformed linear mixed models (FaST-LMM) demonstrated the most superior efficiency, yielding more results with the minimal false positives. We discovered that PUS7, FBXO11, FOXN2, MSH6, and SLC4A4 were associated with BWB. RAG2, and TMEFF2 were candidate genes for BW1, and STARD13, Klotho, ZAR1L are likely candidates for BW3 and BW5. PLXNC1, ATP1A1, CD58, FRYL, OCIAD1, and OCIAD2 were linked to BW7. These findings provide a genetic reference for the selection and breeding of Jinling White ducks, while also deepened our understanding of Growth and development phenotypic in ducks.
Mapping-out baseline physiological muscle parameters with their metabolic blueprint across multiple archetype equine breeds, will contribute to better understanding their functionality, even across species.
Aims: 1) to map out and compare the baseline fiber type composition, fiber type and mean fiber cross-sectional area (fCSA, mfCSA) and metabolic blueprint of three muscles in 3 different breeds 2) to study possible associations between differences in histomorphological parameters and baseline metabolism.
Methods: Muscle biopsies [m. pectoralis (PM), m. vastus lateralis (VL) and m. semitendinosus (ST)] were harvested of 7 untrained Friesians, 12 Standardbred and 4 Warmblood mares. Untargeted metabolomics was performed on the VL and PM of Friesian and Warmblood horses and the VL of Standardbreds using UHPLC/MS/MS and GC/MS. Breed effect on fiber type percentage and fCSA and mfCSA was tested with Kruskal-Wallis. Breeds were compared with Wilcoxon rank-sum test, with Bonferroni correction. Spearman correlation explored the association between the metabolic blueprint and morphometric parameters.
Results: The ST was least and the VL most discriminative across breeds. In Standardbreds, a significantly higher proportion of type IIA fibers was represented in PM and VL. Friesians showed a significantly higher representation of type IIX fibers in the PM. No significant differences in fCSA were present across breeds. A significantly larger mfCSA was seen in the VL of Standardbreds. Lipid and nucleotide super pathways were significantly more upregulated in Friesians, with increased activity of short and medium-chain acylcarnitines together with increased abundance of long chain and polyunsaturated fatty acids. Standardbreds showed highly active xenobiotic pathways and high activity of long and very long chain acylcarnitines. Amino acid metabolism was similar across breeds, with branched and aromatic amino acid sub-pathways being highly active in Friesians. Carbohydrate, amino acid and nucleotide super pathways and carnitine metabolism showed higher activity in Warmbloods compared to Standardbreds.
Conclusion: Results show important metabolic differences between equine breeds for lipid, amino acid, nucleotide and carbohydrate metabolism and in that order. Mapping the metabolic profile together with morphometric parameters provides trainers, owners and researchers with crucial information to develop future strategies with respect to customized training and dietary regimens to reach full potential in optimal welfare.
TNNI1 encodes the slow skeletal muscle isoform of troponin I. In the present study, the basic characteristic and expressing profile of the TNNI1 gene was first explored in Gaoyou ducks. Full-length TNNI1 cDNA of Gaoyou duck was obtained using reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cDNA consisted of a 57-base pair (bp) 5′UTR, a 345-bp 3′UTR, and a 564-bp open reading frame. The predicted protein was predicted to be hydrophilic, nonsecretory protein and contained 17 phosphorylation sites. Multiple alignments and phylogenetic tree analyses showed that the predicted protein was relatively conserved in avian. TNNI1 mRNA could be detected in every tissue analyzed at 70 days of age, and the muscle tissues had relatively high expression level, with the highest level seen in leg muscle. The TNNI1 gene was differentially expressed in the breast muscle and leg muscle during embryonic and posthatching development. Our findings reveal the sequence characterization and expression patterns of the TNNI1 gene, which may provide correlative evidence that TNNI1 gene plays an important role in duck muscle fiber development and meat quality.
Satellite cells derived from fast and slow muscles have been shown to adopt contractile and metabolic properties of their parent muscle. Mouse muscle shows less distinctive fiber-type profiles than rat or rabbit muscle. Therefore, in this study we sought to determine whether three-dimensional muscle constructs engineered from slow soleus (SOL) and fast tibialis anterior (TA) from mice would adopt the contractile and metabolic properties of their parent muscle. Time-to-peak tension (TPT) and half-relaxation time (1/2RT) was significantly slower in SOL constructs. In agreement with TPT, TA constructs contained significantly higher levels of fast myosin heavy chain (MHC) and fast troponin C, I, and T isoforms. Fast SERCA protein, both slow and fast calsequestrin isoforms and parvalbumin were found at higher levels in TA constructs. SOL constructs were more fatigue resistant and contained higher levels of the mitochondrial proteins SDH and ATP synthase and the fatty acid transporter CPT-1. SOL constructs contained lower levels of the glycolytic enzyme phosphofructokinase but higher levels of the β-oxidation enzymes LCAD and VLCAD suggesting greater fat oxidation. Despite no changes in PGC-1α protein, SOL constructs contained higher levels of SIRT1 and PRC. TA constructs contained higher levels of the slow-fiber program repressor SOX6 and the six transcriptional complex (STC) proteins Eya1and Six4 which may underlie the higher in fast-fiber and lower slow-fiber program proteins. Overall, we have found that muscles engineered from predominantly slow and fast mouse muscle retain contractile and metabolic properties of their native muscle. J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
Background:
Among the complexities of skeletal muscle differentiation is a temporal distinction in the onset of expression of different lineage-specific genes. The lineage-determining factor MyoD is bound to myogenic genes at the onset of differentiation whether gene activation is immediate or delayed. How temporal regulation of differentiation-specific genes is established remains unclear.
Results:
Using embryonic tissue, we addressed the molecular differences in the organization of the myogenin and muscle creatine kinase (MCK) gene promoters by examining regulatory factor binding as a function of both time and spatial organization during somitogenesis. At the myogenin promoter, binding of the homeodomain factor Pbx1 coincided with H3 hyperacetylation and was followed by binding of co-activators that modulate chromatin structure. MyoD and myogenin binding occurred subsequently, demonstrating that Pbx1 facilitates chromatin remodeling and modification before myogenic regulatory factor binding. At the same time, the MCK promoter was bound by HDAC2 and MyoD, and activating histone marks were largely absent. The association of HDAC2 and MyoD was confirmed by co-immunoprecipitation, proximity ligation assay (PLA), and sequential ChIP.
Conclusions:
MyoD differentially promotes activated and repressed chromatin structures at myogenic genes early after the onset of skeletal muscle differentiation in the developing mouse embryo.
Skeletal muscle comprises a heterogeneous population of fibers with important physiological differences. Fast fibers are glycolytic and fatigue rapidly. Slow fibers utilize oxidative metabolism and are fatigue resistant. Muscle diseases such as sarcopenia and atrophy selectively affect fast fibers, but the molecular mechanisms regulating fiber type-specific gene expression remain incompletely understood. Here, we show that the transcription factor NFATc1 controls fiber type composition and is required for fast-to-slow fiber type switching in response to exercise in vivo. Moreover, MyoD is a crucial transcriptional effector of the fast fiber phenotype, and we show that NFATc1 inhibits MyoD-dependent fast fiber gene promoters by physically interacting with the N-terminal activation domain of MyoD and blocking recruitment of the essential transcriptional coactivator p300. These studies establish a molecular mechanism for fiber type switching through direct inhibition of MyoD to control the opposing roles of MyoD and NFATc1 in fast versus slow fiber phenotypes.
The important roles of myogenic regulatory factors (MRF) in mammalian skeletal myogenesis have been well studied, but few
equivalent studies have been performed in poultry. The expression pattern of MRF during the embryonic development of skeletal
muscle in ducks remains unknown. In this study, we identified Myf5, Myf6, MyoD, and myogenin genes in Jinding ducks (Anas platyrhynchos domestica) and quantified their expression levels in breast muscle (BM) and leg muscle (LM) at embryonic d 13, 17, 21, 25, and 27 by
real-time reverse-transcription PCR. Body weight and muscle weight show different developmental patterns. The MRF genes were
expressed in both BM and LM, but with different expression patterns. The MyoD gene showed lower expression levels in BM before
embryonic d 21 compared with LM, whereas the opposite pattern was found later. The higher expression level of MyoD, as well
its lagged expression pattern in BM, suggest that the MyoD gene may be involved in maintaining the development of different
muscles. Correlation analysis showed that myogenin gene expression levels were significantly negatively correlated with BW
and muscle weight in both BM and LM (P < 0.001), and MyoD and Myf6 gene expression levels were more strongly correlated with muscle weight in LM than in BM. The
results of this study provide novel evidence for MRF expression in ducks in embryonic stage- and skeletal muscle-dependent
manners, and provide a foundation for understanding the molecular control of skeletal muscle growth in duck breeds.
This book describes the development, growth and adaptation of livestock muscle tissue and contains 18 chapters divided into physiology, genetics and meat quality sections. The physiology section contains chapters on the mechanism of muscle fibre development in the fetus and the importance of high muscle fibre numbers for muscle mass and meat quality (1); muscle fibre type identification and characterization in livestock (2); manipulation of muscle fibre number during prenatal development (3); the effect of growth and exercise on muscle characteristics in relation to meat quality (4); implications of nutrition, hormone receptor expression and gene interactions for muscle development and disease (5); the impact of minerals and micronutrients on growth control (6); significance of exercise and thyroid hormones for development and performance (7); local and systemic regulation of muscle growth (8) and proteolytic systems and regulation of muscle remodelling and breakdown (9). The genetics section contains chapters on the muscle regulatory factors gene family in relation to meat production (10); the muscle transcriptome (11); genome analysis of quantitative trait loci for muscle tissue development and meat quality (12); functional genomics and proteomics in relation to muscle tissue (13); role of myostatin in muscle growth (14) and the genetics, physiology and meat quality aspects of the Callipyge mutation for sheep muscular hypertrophy (15). The meat quality section contains chapters on the genetic control of intramuscular fat accretion (16); postmortem muscle proteolysis and meat tenderness (17) and the water holding capacity of meat (18). Each chapter ends with a list of references and an index is located at the end of the book. This book will be of value for those interested in skeletal muscle biology and meat quality.
Controversies surrounding the efficacy of surgical sealants against alveolar air leaks (AAL) in lung surgery abound in the literature. We sought to test the sealing efficacy of a novel synthetic sealant, TissuePatch™ in an in vitro lung model.
The lower lobe of freshly excised swine lung (n = 10) was intubated and ventilated. A superficial parenchymal defect (40 × 25 mm) was created, followed by AAL assessment. After sealant application, AAL was assessed again until burst failure occurred. The length of defect was recorded to evaluate the elasticity of the sealant.
Superficial parenchymal defects resulted in AAL increasing disproportionally with ascending maximal inspiratory pressure (Pmax). Multiple linear regression analysis revealed strong correlation between AAL and Pmax, compliance, resistance. After sealant application, AAL was sealed in all ten tests at an inspired tidal volume (TVi) of 400 ml, in nine tests at TVi = 500 ml, in seven at TVi = 600 ml and in five at TVi = 700 ml. The mean burst pressure was 42 ± 9 mBar. Adhesive and cohesive sealant failures were found in six and three tests respectively. The length of defect before sealant failure was 8.9 ± 4.9% larger than that at TVi = 400 ml, demonstrating an adequate elasticity of this sealant film.
TissuePatch™ may be a reliable sealant for alternative or adjunctive treatment for repair of superficial parenchymal defects in lung surgery. The clinical benefits of this sealant should be confirmed by prospective, randomised controlled clinical trials.
Die Wirksamkeit von chirurgischen Klebstoffen zur Prävention von alveolo-pleuralem Luftleck (APL) ist trotz zunehmenden klinischen Anwendungen in Lungenchirurgie immer noch kontrovers diskutiert. Wir evaluierten die Abdichtungswirksamkeit von einem neuartigen synthetischen Kleber, TissuePatch™ mittels eines in vitro Lungenmodels.
Der Unterlappen von frisch entnommenen Schweinlungen (n = 10) wurde intubiert und beatmet. Eine pleurale Läsion (40 × 25 mm) wurde erstellt und APL mit steigendem inspiratorischem Tidalvolumen (TVi) untersucht. Nach Applikation von TissuePatch™ wurde APL auf die gleiche Weise gemessen bis zur Auftritt von Kleberbruch. Zur Untersuchung der Elastizität des Klebers wurde die Länge der pleuralen Läsion gemessen.
Pleurale Läsion führte bei aufsteigendem maximalem inspiratorischem Druck (Pmax) zu überproportionalem Anstieg von APL. Multiple lineare Regressionsanalyse ergab eine starke Korrelation zwischen APL und Pmax, Lungencompliance sowie Widerstand. Nach der Applikation von Klebstoff wurde APL bei TVi = 400 ml in allen zehn Testen versiegelt, bei TVi = 500 ml in neun Testen, bei TVi = 600 ml in sieben und bei TVi = 700 ml in fünf Testen. Der mittlere Pmax, der zu Kleberbruch führte, betrug 42 ± 9 mBar. Bei den Versuchen wurden adhäsiver und kohäsiver Kleberbruch in jeweils sechs und drei Testen gefunden. Die Länge der pleuralen Läsion vor dem Kleberbruch war 8,9 ± 4,9% größer als die bei TVi = 400 ml.
Unsere Versuche zeigten eine zuverlässige Versiegelung von TissuePatch™ unter mechanischer Ventilation. Die klinische Nützlichkeit vom Kleber als unterstützende Maßnahme zur Prävention von alveolo-pleuralem Luftleck in Lungenchirurgie sollte durch prospektive, randomisierte kontrollierte klinische Studien bestätigt werden.
Enhancing meat production by genetic selection for growth has already produced 5-week-old broilers weighing more than 2 kg. As growth performance characteristics continue to improve and the time it takes to achieve market size decreases, the period of embryonic development becomes a greater proportion of the bird's life. Therefore, in parallel to genetic selection, other approaches, such as environmental manipulations in the embryo or in the early days posthatch, are becoming more relevant for increasing muscle growth and meat production.Recently, we have shown that nutritional treatments, i.e., providing feed immediately posthatch, or environmental treatments, such as heat conditioning or monochromatic green-light illumination during the first days posthatch, increase muscle growth and breast muscle weight at marketing day. In all cases, the increase in muscle growth was due to changes at the cellular and molecular levels leading to increased satellite cell proliferation and differentiation.The significant effects on muscle growth resulting from the treatments in the first days posthatch raised the hypothesis that muscle growth could be affected during the embryonic development. In experiments in which eggs were illuminated under monochromatic green light from embryonic day 5 (E5), there was a positive effect on embryo development and posthatch muscle growth. Further studies revealed that this enhanced muscle weight was due to increased satellite cell number and fiber synchronization during early days posthatch. Thermal manipulation at 38.5°C from E16 to E18 for 3 h/day had a delayed effect on satellite-cell proliferation and differentiation, resulting in enhanced hypertrophy of myofibers at market age.
Due to selection for increased body weight modern broilers are 3-4 times heavier as compared with chickens of the laying type. The muscle mass is mainly determined by the total number of muscle fibres (hyperplasia), their thickness (hypertrophy) and different fibre types. Hyperplasia occurs during either embryogenesis or the early posthatching period. Skeletal muscles originate from the dermatomyotome, which differentiates into four myogenic cell populations: myotomal cells, embryonic myoblasts, fetal myoblasts and satellite cells; the latter are the adult myoblasts, present within adult skeletal muscles to serve as a cell source for both muscle regeneration and self-renewal. Pax3 keeps migrated precursor cells non-differentiated, thereby controlling transcription of the MyoD gene, whereas Pax7 is a significant regulator of the satellite cell population. Manipulation of temperature and light quality and quantity have been proposed as methods of both pre- and postnatal myogenesis stimulation. Being thermogenic stimulants, both thyroid and adrenal hormones substantially stimulate metabolism. Short-term exposure of embryos to increased temperature between days 16 and 18 of incubation directly influences the proliferation and differentiation of muscle fibres, which manifest themselves in increased hyperplasia. Ultraviolet radiation is an effective means for disinfection of hatching eggs, resulting in a change of embryonic mortality rate during breeding. Especially, green light influences both body weight and the satellite cell number in the first days posthatch, thereby enhancing the growth of embryos, and causing a significant increase in both muscle and body weight. In ovo green stimulation probably enhances the proliferation and differentiation of myoblasts, subsequently causing an increase in muscle weight. The present paper highlights the possibilities of enhancing growth and development of skeletal muscles in birds by manipulation of many aspects of their regulation, thereby contributing to a further increase in production efficiency.
Isozymes of myosin have been localized with respect to individual fibers in differentiating skeletal muscles of the rat and chicken using immunocytochemistry. The myosin light chain pattern has been analyzed in the same muscles by two-dimensional PAGE. In the muscles of both species, the response to antibodies against fast and slow adult myosin is consistent with the speed of contraction of the muscle. During early development, when speed of contraction is slow in future fast and slow muscles, all the fibers react strongly with anti-slow as well as with anti-fast myosin. As adult contractile properties are acquired, the fibers react with antibodies specific for either fast or slow myosin, but few fibers react with both antibodies. The myosin light chain pattern slow shows a change with development: the initial light chains (LC) are principally of the fast type, LC1(f), and LC2(f), independent of whether the embryonic muscle is destined to become a fast or a slow muscle in the adult. The LC3(f), light chain does not appear in significant amounts until after birth, in agreement with earlier reports. The predominance of fast light chains during early stages of development is especially evident in the rat soleus and chicken ALD, both slow muscles, in which LC1(f), is gradually replaced by the slow light chain, LC1(s), as development proceeds. Other features of the light chain pattern include an "embryonic" light chain in fetal and neonatal muscles of the rat, as originally demonstrated by R.G. Whalen, G.S. Butler- Browne, and F. Gros. (1978. J. Mol. Biol. 126:415-431.); and the presence of approximately 10 percent slow light chains in embryonic pectoralis, a fast white muscle in the adult chicken. The response of differentiating muscle fibers to anti-slow myosin antibody cannot, however, be ascribed solely to the presence of slow light chains, since antibody specific for the slow heavy chain continues to react with all the fibers. We conclude that during early development, the myosin consists of a population of molecules in which the heavy chain can be associated with a fast, slow, or embryonic light chain. Biochemical analysis has shown that this embryonic heavy chain (or chains) is distinct from adult fast or slow myosin (R.G. Whalen, K. Schwartz, P. Bouveret, S.M. Sell, and F. Gros. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:5197-5201. J.I. Rushbrook, and A. Stracher. 1979. Proc Natl. Acad. Sci. U.S.A. 76:4331-4334. P.A. Benfield, S. Lowey, and D.D. LeBlanc. 1981. Biophys. J. 33(2, Pt. 2):243a[Abstr.]). Embryonic myosin, therefore, constitutes a unique class of molecules, whose synthesis ceases before the muscle differentiates into an adult pattern of fiber types.
The Silences of the Archives, the Reknown of the Story.
The Martin Guerre affair has been told many times since Jean de Coras and Guillaume Lesueur published their stories in 1561. It is in many ways a perfect intrigue with uncanny resemblance, persuasive deception and a surprizing end when the two Martin stood face to face, memory to memory, before captivated judges and a guilty feeling Bertrande de Rols. The historian wanted to go beyond the known story in order to discover the world of the heroes. This research led to disappointments and surprizes as documents were discovered concerning the environment of Artigat’s inhabitants and bearing directly on the main characters thanks to notarial contracts. Along the way, study of the works of Coras and Lesueur took a new direction. Coming back to the affair a quarter century later did not result in finding new documents (some are perhaps still buried in Spanish archives), but by going back over her tracks, the historian could only be struck by the silences of the archives that refuse to reveal their secrets and, at the same time, by the possible openings they suggest, by the intuition that almost invisible threads link here and there characters and events.
Muscle regulatory factors activate myogenesis in all vertebrates, but their role has been studied in great detail only in the mouse embryo, where all but myogenin--Myod, Myf5 and Mrf4--are sufficient to activate (albeit not completely) skeletal myogenesis. In the zebrafish embryo, myod and myf5 are required for induction of myogenesis because their simultaneous ablation prevents muscle development. Here we show that mrf4 but not myog can fully rescue myogenesis in the myod/myf5 double morphant via a selective and robust activation of myod, in keeping with its chromatin-remodelling function in vitro. Rescue does not happen spontaneously, because the gene, unlike that in the mouse embryo, is expressed only at the onset of muscle differentiation, Moreover, because of the transient nature of morpholino inhibition, we were able to investigate how myogenesis occurs in the absence of a myotome. We report that in the complete absence of a myotome, subsequent myogenesis is abolished, whereas myogenesis does proceed, albeit abnormally, when the morpholino inhibition was not complete. Therefore our data also show that the early myotome is essential for subsequent skeletal muscle differentiation and patterning in the zebrafish.
The molecular basis of skeletal muscle lineage determination was investigated by analyzing DNA control elements that regulate
the myogenic determination gene myoD. A distal enhancer was identified that positively regulates expression of the human myoD
gene. The myoD enhancer and promoter were active in myogenic and several nonmyogenic cell lines. In transgenic mouse embryos,
however, the myoD enhancer and promoter together directed expression of a lacZ transgene specifically to the skeletal muscle
lineage. These data suggest that during development myoD is regulated by mechanisms that restrict accessibility of myoD control
elements to positive trans-acting factors.
This article reviews the complexity, expression, genetics, regulation, function, and evolution of the avian myosin heavy chain (MyHC). The majority of pertinent studies thus far published have focussed on domestic chicken and, to a much lesser extent, Japanese quail. Where possible, information available about wild species has also been incorporated into this review. While studies of additional species might modify current interpretations, existing data suggest that some fundamental properties of myosin proteins and genes in birds are unique among higher vertebrates. We compare the characteristics of myosins in birds to those of mammals, and discuss potential molecular mechanisms and evolutionary forces that may explain how avian MyHCs acquired these properties.
Age-related changes in satellite cell proliferation and differentiation during rapid growth of porcine skeletal muscle were examined. Satellite cells were isolated from hindlimb muscles of pigs at 1, 7, 14, and 21 wk of age (4 animals/age group). Satellite cells were separated from cellular debris by using Percoll gradient centrifugation and were adsorbed to glass coverslips for fluorescent immunostaining. Positive staining for neural cell adhesion molecule (NCAM) distinguished satellite cells from nonmyogenic cells. The proportion of NCAM-positive cells (satellite cells) in isolates decreased from 1 to 7 wk of age. Greater than 77% of NCAM-positive cells were proliferating cell nuclear antigen positive at all ages studied. Myogenin-positive satellite cells decreased from 30% at 1 wk to 14% at 7 wk of age and remained at constant levels thereafter. These data indicate that a high percentage of satellite cells remain proliferative during rapid postnatal muscle growth. The reduced proportion of myogenin-positive cells during growth may reflect a decrease in the proportion of differentiating satellite cells or accelerated incorporation of myogenin-positive cells into myofibers.
Embryological and genetic studies of mouse, bird, zebrafish, and frog embryos are providing new insights into the regulatory functions of the myogenic regulatory factors, MyoD, Myf5, Myogenin, and MRF4, and the transcriptional and signaling mechanisms that control their expression during the specification and differentiation of muscle progenitors. Myf5 and MyoD genes have genetically redundant, but developmentally distinct regulatory functions in the specification and the differentiation of somite and head muscle progenitor lineages. Myogenin and MRF4 have later functions in muscle differentiation, and Pax and Hox genes coordinate the migration and specification of somite progenitors at sites of hypaxial and limb muscle formation in the embryo body. Transcription enhancers that control Myf5 and MyoD activation in muscle progenitors and maintain their expression during muscle differentiation have been identified by transgenic analysis. In epaxial, hypaxial, limb, and head muscle progenitors, Myf5 is controlled by lineage-specific transcription enhancers, providing evidence that multiple mechanisms control progenitor specification at different sites of myogenesis in the embryo. Developmental signaling ligands and their signal transduction effectors function both interactively and independently to control Myf5 and MyoD activation in muscle progenitor lineages, likely through direct regulation of their transcription enhancers. Future investigations of the signaling and transcriptional mechanisms that control Myf5 and MyoD in the muscle progenitor lineages of different vertebrate embryos can be expected to provide a detailed understanding of the developmental and evolutionary mechanisms for anatomical muscles formation in vertebrates. This knowledge will be a foundation for development of stem cell therapies to repair diseased and damaged muscles.
Muscle development at 20 and 25 d of incubation was studied in a randombred control line (RBC2), a subline (F) of RBC2 selected only for increased 16-wk BW, a commercial sire line (B), and reciprocal crosses of the F and B lines. Muscle samples from three males and three females of each genetic group were collected in such a manner to avoid contraction. After fixing, the muscles were stained with hematoxylin and eosin, measurements of muscle fiber width, muscle fiber bundle length and width, number of fibers within a 15.6 microm2 area, and extracellular matrix perimysial (PW) and endomysial (EW) width were taken with an Olympus XI 70 microscope equipped with an Olympus Magna Fire digital camera linked to Image Pro software. From each slide, 20 measurements were taken for each characteristic analyzed. In most of the muscle traits measured, additive genetic variation, as indicated by line differences, occurred when the RBC2 line was included in the comparison of pure lines. However, when only the B and F lines were compared, line differences were less frequent. In comparisons of the B and F lines and their reciprocal crosses, heterosis, as measured by contrasts of the average of the pure lines and the average of the reciprocal crosses, was an important source of variation for individual fiber measurements (negative) and extracellular space (positive) at 20 d of incubation but was less important at 25 d of incubation. No significant interactions between genetic group and sex were noted at 20 d of incubation, but such interactions were frequent at 25 d of incubation. These results suggest that muscle organizational differences between the two sexes begin to occur between these two ages and are not the same for different genetic groups.
In farm animals (bovine, ovine, swine, rabbit and poultry), muscle fibre characteristics play a key role in meat quality. The present review summarises the knowledge on muscle fibre characteristics and ontogenesis in these species. Myofibre ontogenesis begins very early during embryonic life, with the appearance of two or three successive waves of myoblasts which constitute the origin of the different types of muscle fibres. In small animals (rodents and poultry), a primary and a secondary generation of fibres arise respectively during the embryonic and foetal stages of development. In the largest species (bovines, sheep, pigs) a third generation arises in the late foetal or early postnatal period. Following these two or three waves of myogenesis, the total number of fibres is fixed. This occurs during foetal life (bovines, ovines, pigs and poultry) or during the first postnatal month in rabbits. Contractile and metabolic differentiation proceed by steps in parallel to myogenesis and are partially linked to each other. In bovines and ovines, the main events occur during foetal life, whereas they occur soon after birth in the pig, poultry and rabbit, but some plasticity remains later in life in all species. This comparative survey shows that the cellular processes of differentiation are comparable between species, while their timing is usually species specific.
The paired-box transcription factor Pax7 plays a critical role in the specification of satellite cells in mouse skeletal muscle. In the present study, the position and number of Pax7-expressing cells found in muscles of growing and adult chickens confirm the presence of this protein in avian satellite cells. The expression pattern of Pax7 protein, along with the muscle regulatory proteins MyoD and myogenin, was additionally elucidated in myogenic cultures and in whole muscle from posthatch chickens. In cultures progressing from proliferation to differentiation, the expression of Pax7 in MyoD+ cells declined as the cells began expressing myogenin, suggesting Pax7 as an early marker for proliferating myoblasts. At all time points, some Pax7+ cells were negative for MyoD, resembling the reserve cell phenotype. Clonal analysis of muscle cell preparations demonstrated that single progenitors can give rise to both differentiating and reserve cells. In muscle tissues, Pax7 protein expression was the strongest by 1 day posthatch, declining on days 3 and 6 to a similar level. In contrast, myogenin expression peaked on day 3 and then dramatically declined. This finding was accompanied by a robust growth in fiber diameter between day 3 and 6. The distinctions in Pax7 and myogenin expression patterns, both in culture and in vivo, indicate that while some of the myoblasts differentiate and fuse into myofibers during early stages of posthatch growth, others retain their reserve cell capacity.
This presentation aims to describe how the basic events in prenatal muscle development and postnatal muscle growth are controlled by the insulin-like growth factor system (IGF). The prenatal events (myogenesis) cover the rate of proliferation, the rate and extent of fusion, and the differentiation of three myoblast populations, giving rise to primary fibers, secondary fibers, and a satellite cell population, respectively. The number of muscle fibers, a key determinant of the postnatal growth rate, is fixed late in gestation. The postnatal events contributing to myofiber hypertrophy comprise satellite cell proliferation and differentiation, and protein turnover. Muscle cell cultures produce IGFs and IGF binding proteins (IGFBPs) in various degrees depending on the origin (species, muscle type) and state of development of these cells, suggesting an autocrine/paracrine mode of action of IGF-related factors. In vivo studies and results based on cell lines or primary cell cultures show that IGF-I and IGF-II stimulate both proliferation and differentiation of myoblasts and satellite cells in a time and concentration-dependent way, via interaction with type I IGF receptors. However, IGF binding proteins (IGFBP) may either inhibit or potentiate the stimulating effects of IGFs on proliferation or differentiation. During postnatal growth in vivo or in fully differentiated muscle cells in culture, IGF-I stimulates the rate of protein synthesis and inhibits the rate of protein degradation, thereby enhancing myofiber hypertrophy. The possible roles and actions of the IGF system in regulating and determining muscle growth as affected by developmental stage and age, muscle type, feeding levels, treatment with growth hormone and selection for growth performance are discussed.
The development and differentiation of distinct cell types is achieved through the sequential expression of subsets of genes; yet, the molecular mechanisms that temporally pattern gene expression remain largely unknown. In skeletal myogenesis, gene expression is initiated by MyoD and includes the expression of specific Mef2 isoforms and activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Here, we show that p38 activity facilitates MyoD and Mef2 binding at a subset of late-activated promoters, and the binding of Mef2D recruits Pol II. Most importantly, expression of late-activated genes can be shifted to the early stages of differentiation by precocious activation of p38 and expression of Mef2D, demonstrating that a MyoD-mediated feed-forward circuit temporally patterns gene expression.
We used a combination of genome-wide and promoter-specific DNA binding and expression analyses to assess the functional roles of Myod and Myog in regulating the program of skeletal muscle gene expression. Our findings indicate that Myod and Myog have distinct regulatory roles at a similar set of target genes. At genes expressed throughout the program of myogenic differentiation, Myod can bind and recruit histone acetyltransferases. At early targets, Myod is sufficient for near full expression, whereas, at late expressed genes, Myod initiates regional histone modification but is not sufficient for gene expression. At these late genes, Myog does not bind efficiently without Myod; however, transcriptional activation requires the combined activity of Myod and Myog. Therefore, the role of Myog in mediating terminal differentiation is, in part, to enhance expression of a subset of genes previously initiated by Myod.
We identify here the multiple epidermal growth factor repeat transmembrane protein Megf10 as a quiescent satellite cell marker that is also expressed in skeletal myoblasts but not in differentiated myofibers. Retroviral expression of Megf10 in myoblasts results in enhanced proliferation and inhibited differentiation. Infected myoblasts that fail to differentiate undergo cell cycle arrest and can reenter the cell cycle upon serum restimulation. Moreover, experimental modulations of Megf10 alter the expression levels of Pax7 and the myogenic regulatory factors. In contrast, Megf10 silencing in activated satellite cells on individual fibers or in cultured myoblasts results in a dramatic reduction in the cell number, caused by myogenin activation and precocious differentiation as well as a depletion of the self-renewing Pax7+/MyoD- population. Additionally, Megf10 silencing in MyoD-/- myoblasts results in down-regulation of Notch signaling components. We conclude that Megf10 represents a novel transmembrane protein that impinges on Notch signaling to regulate the satellite cell population balance between proliferation and differentiation.
Intensive selection conducted within closed populations has led to the creation of specialized chicken strains that differ significantly in meat yield and reproduction performance. The effect of the selection conducted on the birds is differentiation identified not only on the molecular but also on the cellular level, among other things in the skeletal muscles. The aim of this study was to compare the structure of chosen homological skeletal muscles from Leghorn chickens (LSL), originating from parent flock, intensively selected for reproductive traits and from conservative flock (G99), unselected for many generations. The structure of musculus pectoralis superficialis and musculus biceps femoris (the thickness of the muscle fibres and the share of the fibre types in the bundle) in 8 and 20 week old chickens was compared. A significant impact of the origin on all examined slaughter parameters was recorded. Body weight before slaughter, carcass weight and the weight of breast and leg muscles in 8 weeks old LSL chicken made up from 60% to about 85% of the respective values in the G99 Leghorn. Lack of red fibres in the breast muscles of all the individuals from the parental flock (LSL) was noted, whereas in 12 individuals (among 24) from the conservative flock (G99), red fibres were observed in this muscle from 2.75% up to 7.09%. White fibres in 8 week old chicks were always thicker, both in pectoralis superficialis and biceps femoris muscle in birds with higher body weight as well as higher weight of breast and legs muscles, i.e. in chicks from conservative flock (G99), P<0.01. However, in 20 week old birds, the diameters of the white fibres were similar in both groups. Also the diameters of the red fibres in musculus biceps femoris in 8 week old chickens were higher in cockerels and pullets from conservative flock (G99).
Unlike the mammalian fetus, whose growth is supported by the sustained provision of maternal nutrients, poultry embryos undergo development in a relatively closed space, and the yolk sac serves as the sole nutrient supply for embryonic development throughout the whole incubation period. To increase our understanding of the muscle developmental patterns in the final stage of incubation and early days posthatching, we used late-term duck embryos and newly hatched ducklings as animal models. Pectoralis muscle samples were collected at 22 days (22E) of incubation, 25 days (25E) of incubation, hatching and day 7 posthatching. The pectoralis muscle mass, muscle fibre bundles and myofibre cross-sectional area showed a marked reduction from 22E to hatching, but they increased dramatically by day 7 posthatching. The mRNA expression of Atrogin-1, a key mediator of the ubiquitin system responsible for protein degradation, increased dramatically with the age of late-term duck embryos, but it decreased by day 7 and reached a very low level. The extent of mRNA expression of FoxO1, one of the transcription factors of the Atrogin-1 gene, exhibited a transient increase at 25E and then decreased from hatching to day 7. The phosphorylated p70 ribosomal protein S6 kinase 1 (S6K1)/S6K1 ratio exhibited a dramatic reduction from 22E to hatching (P < 0.05) and then increased by day 7. The results of the present study indicated that there was a developmental transition of pectoralis muscle from atrophy in late-term duck embryos to hypertrophy in neonates.
While the existence of post-hatch and adult myosin heavy chain isoforms in the large, avian type IIB pectoralis major muscle has been clearly established, the number and nature of fast myosin heavy chains during in ovo development and the perihatch period have not been resolved. In the present study, developmental fast heavy chain proteins purified by high resolution anion-exchange have been characterized by sequence analysis of a unique CNBr peptide and by complementary mRNA analysis. The four proteins present at 15/16 days in ovo are shown to differ uniquely in primary structure. They correlate with heavy chains II, IV, VI and VII, characterized recently as major or minor species in adult fast muscles using similar methods. These four heavy chains are expressed in a time-dependent fashion from 8 to 16 days in ovo. At the mRNA level, heavy chain VI predominates until 12 days in ovo. Heavy chain IV mRNA is upregulated dramatically at 16 days in ovo preparatory to its protein's predominance in the peri-hatch period. Heavy chains II, IV and V (the post-hatch isoform which replaces heavy chain IV) have major roles in adult fast muscles.
In pigs, myogenesis is a biphasic phenomenon with the formation of primary and secondary fibres. Hyperplasia was reported to be accomplished around 90 days of gestation. However, some studies suggest a substantial increase in the total fibre number (TFN) from birth to weaning by counting fibre number in the muscle cross sections. The aim of this study was to establish in which way TFN increases after birth and whether this increase is imputable to new (tertiary) myofibres and/or fibre elongation. The semitendinosus muscle of 128 piglets was examined at days 1 (n = 63), 7 (n = 12), 21 (n = 12), and 28 (n = 41) of age. TFN was increased at days 7, 21 and 28 of age when compared with day 1 (P < 0.01). From day 1 to 28, TFN increased from 463 × 10(3) to 825 × 10(3). Microscopy of longitudinal and transversal serial sections revealed that at day 7 of age very small fibres expressing the embryonic myosin heavy chain (MyHC) isoform were apparent all over the muscle. In addition, intrafascicular terminations of normal-sized fibres expressed the embryonic MyHC isoform. These data suggest that the TFN in the pig muscle is not fixed at birth and its postnatal increase may be related to both elongation of existing muscle fibres and genesis of tertiary myofibres, mainly between birth and 3 weeks of age.
In order to investigate the developmental differences between the duck breast muscle and leg muscle tissues during the embryonic stage to neonatal stages, as well as the expression profile of MyoD between the two muscle tissues, the morphologic characteristics in the two muscle tissues during duck embryo stages at E14, E18, E22, E27 and D7 were compared through the muscle paraffin sections. The coding domain sequence of duck MyoD gene was cloned, and then the expression of MyoD in duck leg muscle and breast muscle during embryo stage on E10, E14, E18, E22, E27 and D7 was detected using qRT-PCR method. Results showed that the developmental status of the duck breast muscle in embryonic phrases lag behind that of leg muscle. The CDS of duck MyoD gene consists of 894 nucleotides, and showed relatively high similarity with the gene of other species. The MyoD mRNA expressed in both kinds of muscle tissues and the expression profile had a similar trend, although the expression level of MyoD in the breast muscle was significantly higher than that in the leg muscle at each developmental stages (p<0.05). Results suggested that MyoD might have potential functions in controlling muscle fiber phenotype during the secondary myogenesis of muscle development. These fundamental works may provide some valuable clues for knowing the roles of MyoD in the myogenesis and the muscle fiber type differentiation in birds.
Enhancing skeletal muscle growth is crucial for animal agriculture because skeletal muscle provides meat for human consumption. An increasing body of evidence shows that the level of maternal nutrition alters fetal skeletal muscle development, with long-term effects on offspring growth and performance. Fetal skeletal muscle development mainly involves myogenesis (i.e., muscle cell development), but also involves adipogenesis (i.e., adipocyte development) and fibrogenesis (i.e., fibroblast development). These tissues in fetal muscle are mainly derived from mesenchymal stem cells (MSC). Shifting the commitment of MSC from myogenesis to adipogenesis increases intramuscular fat (i.e., marbling), improving the quality grade of meats. Strong experimental evidence indicates that Wingless and Int (Wnt)/beta-catenin signaling regulates MSC differentiation. Upregulation of Wnt/beta-catenin promotes myogenesis, and downregulation enhances adipogenesis. A lack of nutrients in early to midgestation reduces the formation of secondary muscle fibers in ruminant animals. Nutrient deficiency during mid- to late gestation decreases the number of intramuscular adipocytes and muscle fiber sizes. Knowledge of this regulatory mechanism will allow the development of strategies to enhance muscle growth and marbling in offspring, especially in the setting of nutrient deficiency.
Myostatin (Mstn) is a secreted growth factor belonging to the tranforming growth factor (TGF)-beta superfamily. Inactivation of murine Mstn by gene targeting, or natural mutation of bovine or human Mstn, induces the double muscling (DM) phenotype. In DM cattle, Mstn deficiency increases fast glycolytic (type IIB) fiber formation in the biceps femoris (BF) muscle. Using Mstn null ((-/-)) mice, we suggest a possible mechanism behind Mstn-mediated fiber-type diversity. Histological analysis revealed increased type IIB fibers with a concomitant decrease in type IIA and type I fibers in the Mstn(-/-) tibialis anterior and BF muscle. Functional electrical stimulation of Mstn(-/-) BF revealed increased fatigue susceptibility, supporting increased type IIB fiber content. Given the role of myocyte enhancer factor 2 (MEF2) in oxidative type I fiber formation, MEF2 levels in Mstn(-/-) tissue were quantified. Results revealed reduced MEF2C protein in Mstn(-/-) muscle and myoblast nuclear extracts. Reduced MEF2-DNA complex was also observed in electrophoretic mobility-shift assay using Mstn(-/-) nuclear extracts. Furthermore, reduced expression of MEF2 downstream target genes MLC1F and calcineurin were found in Mstn(-/-) muscle. Conversely, Mstn addition was sufficient to directly upregulate MLC promoter-enhancer activity in cultured myoblasts. Since high MyoD levels are seen in fast fibers, we analyzed MyoD levels in the muscle. In contrast to MEF2C, MyoD levels were increased in Mstn(-/-) muscle. Together, these results suggest that while Mstn positively regulates MEF2C levels, it negatively regulates MyoD expression in muscle. We propose that Mstn could regulate fiber-type composition by regulating the expression of MEF2C and MyoD during myogenesis.
The myogenic basic HLH transcription factor family of genes, composed of MyoD, myogenin, Myf-5, and Myf-6, are thought to regulate skeletal muscle differentiation. To understand the role of MyoD in myogenesis, we have introduced a null mutation of MyoD into the germline of mice. Surprisingly, mice lacking MyoD are viable and fertile. Histological examination of skeletal muscle failed to reveal any morphological abnormalities in these mice. Furthermore, Northern analysis revealed normal levels of skeletal muscle-specific mRNAs. Significantly, Myf-5 mRNA levels are elevated in postnatal mutant mice. Normally, Myf-5 expression becomes markedly reduced at day 12 of gestation when MyoD mRNA first appears. This suggests that Myf-5 expression is repressed by MyoD. Our results indicate that MyoD is dispensable for skeletal muscle development in mice, revealing some degree of functional redundancy in the control of the skeletal myogenic developmental program.
Immunohistochemistry was used to examine the expression of embryonic, slow, and neonatal isoforms of myosin heavy chain in muscle fibers of the embryonic rat hindlimb. While the embryonic isoform is present in every fiber throughout prenatal development, by the time of birth the expression of the slow and neonatal isoforms occurs, for the most part, in separate, complementary populations of fibers. The pattern of slow and neonatal expression is highly stereotyped in individual muscles and mirrors the distribution of slow and fast fibers found in the adult. This pattern is not present at the early stages of myogenesis but unfolds gradually as different generations of fibers are added. As has been noted by previous investigators (e.g., Narusawa et al., 1987, J. Cell Biol. 104, 447-459), all of the earliest generation (primary) muscle fibers initially express the slow isoform but some of these primary fibers later lose this expression. In this study we show that loss of slow myosin in these fibers is accompanied by the expression of neonatal myosin. This switch in isoform expression occurs in all primary fibers located in specific regions of particular muscles. However, in other muscles primary fibers which retain their slow expression are extensively intermixed with those that switch to neonatal expression. Later generated (secondary) muscle fibers, which are interspersed among the primary fibers, express neonatal myosin, although a few of them in stereotyped locations later switch from neonatal to slow myosin expression. Many of the observed changes in myosin expression occur coincidentally with the arrival of axons in the limb or the invasion of axons into individual muscles. Thus, although both fiber birth date and intramuscular position are grossly predictive of fiber fate, neither factor is sufficient to account for the final pattern of fiber types seen in the rat hindlimb. The possibility that fiber diversification is dependent upon innervation is tested in the accompanying paper (K. Condon, L. Silberstein, H.M. Blau, and W.J. Thompson, 1990, Dev. Biol. 138, 275-295).
A line of Japanese quail selected for high body weight at 4 weeks of age (P line) was compared to an unselected control (C) line at 10, 23 and 56 days of age. The increase in the weight of the pectoralis major and supracoracoideus muscles in the P line was paralleled by an increase in total DNA, RNA and protein content of the muscles when compared to comparable age C line. DNA, RNA and protein concentrations and RNA/DNA and protein/DNA ratios were similar between lines within age. Greater muscle mass in the P line was accomplished primarily through an increase in the total number of muscle nuclei rather than through an increase in DNA unit size. Ca2+-activated myosin ATPase, total phosphorylase and succinic dehydrogenase enzyme activities were similar between lines and across ages in the pectoralis major and supracoracoideus muscles. The semimembranosus muscle possessed 59% more alpha fibers and only 7% more beta fibers in the P line when compared to the C line. Semimembranosus alpha fiber diameters were not significantly different between lines within age, while beta fiber diameters were significantly greater in the P line. Estimated beta fiber contribution to total semimembranosus muscle cross sectional area revealed no significant difference between lines within age. There was a significant increase in the length of the femur and humerus in the P line when compared to C line within age, indicating that some of the increased muscle weight of the P line quail was due to an increase in muscle length in addition to an increase in muscle cross-sectional area.
The objective of the research described herein was to describe the profile of histochemically determined myofiber types in serratus ventralis thoracis of the sheep at various stages of postnatal development. This muscle acts to suspend the trunk and to pull the anterior limbs back during locomotion. The results obtained allow comparison with other results in the literature on muscles with different functional demands of movement and postural activity. Three sheep were sacrificed at each of the following ages: birth, 2, 4, 8, 12 and 16 wk. One 52-mo-old sheep was used. The muscle was processed histochemically for a series of enzyme activities including myosin adenosine triphosphatase (ATPase). Type I myofibers reacted strongly for acid-stable myosin ATPase and negatively for alkali-stable ATPase. Type II myofibers showed the opposite reaction pattern. Various subtypes were classified on the basis of intermediate reaction patterns and on the basis of enzyme activities other than ATPase. Type II myofibers decreased greatly in proportion from birth to 4 wk of age and were essentially unchanged during further growth of the animal. Type I myofibers increased in proportion from birth to 4 wk, increased slightly from 4 to 12 wk and then underwent little further change. Intermediate types changed little from birth to 4 wk and decreased thereafter. Type II myofibers were greater in proportion than type I and intermediate myofibers were always lowest, regardless of age of animal.
Proportions of myofiber types, radial growth (diameter) of myofibers and apparent myofiber number were measured at the midsection of the sartorius muscle of broiler- and layer-type chickens. Broiler birds grew mor rapidly than layer birds so that in comparisons at equal age, broilers had heavier body and muscle weights, larger diameter type I and II myofibers and greater apparent myofiber number. The proportion of type II red myofibers decreased and that of type II white myofibers increased during growth. These changes occurred at a younger age in broiler-type birds. At equal body weights, however, broiler- and layer-type birds had similar proportions of the various myofiber types, which indicates that development of myofiber types is affected by functional demands on skeletal muscle related to increasing body weight. At equal body weights, broilers had larger diameter type II myofibers than layers and had a more rapid rate of type II myofiber radial hypertrophy during growth. In contrast, layer-type birds had larger type I myofibers than did broilers and the rate of radial growth was similar between breeds. Apparent myofiber number per unit of body weight increased during growth at a similar rate in the two types of birds but broilers had greater numbers of myofibers. It was concluded that more rapid growth and greater muscularity of broiler-type birds are caused by more rapid myofiber hypertrophy and the presence of more myofibers. It is suggested that selection for growth and muscularity favors factors that promote selective radial hypertrophy of type II myofibers as seen in broiler-type chickens.
Quantitative (muscle fibre number and cross-sectional areas) and qualitative (myosin isoforms and metabolic enzyme activities) characteristics of two muscles, M. pectoralis major and M. anterior latissimus dorsi, were compared among male chickens of two lines during growth from hatching to adulthood. The lines were derived from a divergent selection based on growth rate. The two muscles were chosen on the basis of their histochemical profile. Pectoralis major muscle contains only fast contracting muscle fibres whereas anterior latissimus dorsi muscle is almost entirely made up with slow contracting fibres. At both ages, the two lines showed similar fibre type distributions. At hatching, fibre cross-sectional areas were equivalent in the two lines, but after the first week, animals from the fast growing line exhibited wider fibre areas, whatever the muscle, than animals from the slow growing line. The total number of fibres in a muscle was found greater in the fast growing line, irrespective of whether it was exactly determined (anterior latissimus dorsi muscle, + 20%) or only estimated (pectoralis major muscle). This number remains constant in the two lines throughout the growth. Myosin isoform profiles and metabolic enzyme activities were similar in the two lines, at both ages, and were in good agreement with the histochemical muscle fibre profiles.
1. Histochemical (fibre type distribution and areas) and biochemical (myosin isoforms) characteristics of three muscles, M. anterior latissimus dorsi, M. pectoralis major and M. sartorius, were compared among male chickens of two lines at 11 and 55 weeks of age. 2. The lines were derived from a divergent selection based on growth rate. Cockerels from the Fast Growing Line (FGL) were 2.3 times heavier than those from the Slow Growing Line (SGL) when 11 weeks old and 1.7 times at 55 weeks of age. The latter age was chosen as representative of the adult stage and the 11-week age because, at this time, FGL cocks weighed as much as SGL cockerels at 55 weeks. 3. At both ages, the two lines showed similar fibre type distributions, but the total number in the ALD muscle, and the size (cross-sectional areas) of fibres in each muscle were higher in the FGL compared with the SGL (14.6% and 33% more at 11 and 55 weeks of age respectively in favour of the FGL birds). 4. The two lines displayed similar myosin isoform patterns when adult muscles were compared (55 weeks). They differed slightly at 11 weeks of age, muscle differentiation being completed only in the FGL. 5. Comparisons of the two lines at the same live weight (i.e. FGL cockerels at 11 weeks of age and SGL cockerels at 55 weeks) showed larger muscle fibres in the SGL and no difference in the isomyosin patterns.
The expression of fast myosin heavy chain (MyHC) genes was examined in vivo during fast skeletal muscle development in the inbred White Leghorn chicken (line 03) and in adult muscles from the genetically related dystrophic White Leghorn chicken (line 433). RNA dotblot and northern hybridization was employed to monitor MyHC transcript levels utilizing specific oligonucleotide probes. The developmental pattern of MyHC gene expression in the pectoralis major (PM) and the gastrocnemius muscles was similar during embryonic development with three embryonic MyHC isoform genes, Cemb1, Cemb2, and Cemb3, sequentially expressed. Following hatching, MyHC expression patterns in each muscle differed. The expression of MyHC genes was also studied in muscle cell cultures derived from 12-day embryonic pectoralis muscles. In vitro, Cvent, Cemb1, and Cemb2 MyHC genes were expressed; however, little if any Cemb3 MyHC gene expression could be detected, even though Cemb3 was the predominant MyHC gene expressed during late embryonic development in vivo. In most adult muscles other than the PM and anterior latissimus dorsi (ALD), the Cemb3 MyHC gene was the major adult MyHC isoform. In addition, two general patterns of expression were identified in fast muscle. The fast muscles of the leg expressed neonatal (Cneo) and Cemb3 MyHC genes, while other fast muscles expressed adult (Cadult) and Cemb3 MyHC genes. MyHC gene expression in adult dystrophic muscles was found to reflect the expression patterns found in corresponding normal muscles during the neonatal or early post-hatch developmental period, providing additional evidence that avian muscular dystrophy inhibits muscle maturation.
In this study, the differentiation of adult and postnatal muscle fibres in sheep longissimus thoracis muscle has been characterized. By using a variety of histochemical methods, we have investigated the m-ATPase and metabolic activities of skeletal muscle fibres in adult sheep and lambs aged between 1 day and 3 months. Types I, IIA, IIB and IIC fibres were identified. The results showed that the interpretation of the fibre type composition depends on the methods used. The findings also revealed that the fibre types IIA and IIB can be separated histochemically in sheep by using the correct m-ATPase technique, even at early stages of postnatal development, and that the origin of the four different fibres of the adult can be traced back to early postnatal stages.
Four major sarcomeric myosin heavy chains (MyHC) (i.e., I, IIa, IIx, and IIb) are expressed in pig skeletal muscle during postnatal development. The objective of the current study was to compare MyHC composition at mRNA and protein levels in LM, a fast-twitch glycolytic muscle, and rhomboideus (RM), a mixed slow- and fast-twitch oxido-glycolytic muscle, between two pig breeds exhibiting dramatic differences in postnatal muscle growth and meat quality. Eight Large White (LW) and eight Meishan (MS) females were fed under the same standard conditions, and slaughtered at an average BW of 62 kg (131 and 142 d in LW and MS pigs, respectively). In addition to conventional fiber typing by histoenzymology, MyHC composition was analyzed by combining immunocytochemistry, in situ hybridization, and a newly developed real-time PCR assay. Enzyme activities of lactate dehydrogenase, citrate synthase, and beta-hydroxy-acyl-CoA-dehydrogenase were used as markers of glycolytic, oxidative and beta-oxidation capacities, respectively. Results showed that conventional fiber typing in three classes by histoenzymology was insufficient in LM. For the first time, four monoclonal antibodies specific of each MyHC isoform, working in immunocytochemistry, were used. Our results are consistent with the sequential I<-->IIa<-->IIx<-->IIb MyHC transition rule. Breed effect on MyHC composition differed between muscle types. In LM of MS pigs, a shift from IIb to IIx, and to a lesser extent, to IIa, occurred without affecting type I MyHC. In RM, where IIb is absent, a shift from IIx to type I occurred, with a slight decrease in the IIa isoform. Effects were very similar at the mRNA and protein levels, suggesting a transcriptional regulation. In both muscles, MS pigs exhibited a decrease in the relative fiber type specific expression of the fastest isoform (i.e., IIb in LM and IIx in RM). The shift toward a slower phenotype in MS pigs was consistent with a less glycolytic and more oxidative metabolism, potentially using more lipids as fuel. A dramatic increase in cross-sectional area of type I fibers in RM (+27%) and a decrease in that of the fastest IIb fibers in LM (-25%) were observed in MS pigs. Overall, interpretation of earlier data regarding muscle fiber type has been flawed by inaccurate fiber typing in most pig skeletal muscles.
Elucidating the molecular pathways linking electrical activity to gene expression is necessary for understanding the effects of exercise on muscle. Fast muscles express higher levels of MyoD and lower levels of myogenin than slow muscles, and we have previously linked myogenin to expression of oxidative enzymes. We here report that in slow muscles, compared with fast, 6 times as much of the MyoD is in an inactive form phosphorylated at T115. In fast muscles, 10 h of slow electrical stimulation had no effect on the total MyoD protein level, but the fraction of phosphorylated MyoD was increased 4-fold. Longer stimulation also decreased the total level of MyoD mRNA and protein, while the level of myogenin protein was increased. Fast patterned stimulation did not have any of these effects. Overexpression of wild type MyoD had variable effects in active slow muscles, but increased expression of fast myosin heavy chain in denervated muscles. In normally active soleus muscles, MyoD mutated at T115 (but not at S200) increased the number of fibres containing fast myosin from 50% to 85% in mice and from 13% to 62% in rats. These data establish de-phosphorylated active MyoD as a link between the pattern of electrical activity and fast fibre type in adult muscles.
Skeletal muscles are classified into fast and slow muscles, which are characterized by the expression of fast-type myosin heavy chains (fMyHCs) or slow-type myosin heavy chains (sMyHCs), respectively. However, the mechanism of subtype determination during muscle fiber regeneration is unclear. We have analyzed whether the type of muscle is determined in the myoblast cells or is controlled by the environment in which the muscle fibers are formed from myoblast cells. When myoblast cells from 7-day-old chick embryo were cultured and formed into muscle fibers, more than half of the fibers produced only fMyHCs, and the remaining fibers produced both fMyHCs and sMyHCs. However, when myoblast cells were cultured in medium supplemented with a small amount of slow muscle extract, the expression of sMyHCs in muscle fibers increased, whereas the expression of fMyHCs increased in the group supplemented with fast muscle extract compared with the control group. The same results were obtained when cloned mouse myoblast cells (C2C12 cells) were cultured and formed into muscle fibers. The data presented here thus show that the subtype differentiation of muscle fiber is controlled by the environment in which the muscle fiber forms.