MicroRNA transcriptome profiles during swine skeletal muscle development

Background: MicroRNA (miR) are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts. To evaluate the role of miR in skeletal muscle of swine, global microRNA abundance was measured at specific developmental stages including proliferating satellite cells, three stages of fetal growth, day-old neonate, and the adult. Results: Twelve potential novel miR were detected that did not match previously reported sequences. In addition, a number of miR previously reported to be expressed in mammalian muscle were detected, having a variety of abundance patterns through muscle development. Muscle-specific miR-206 was nearly absent in proliferating satellite cells in culture, but was the highest abundant miR at other time points evaluated. In addition, miR-1 was moderately abundant throughout developmental stages with highest abundance in the adult. In contrast, miR-133 was moderately abundant in adult muscle and either not detectable or lowly abundant throughout fetal and neonate development. Changes in abundance of ubiquitously expressed miR were also observed. MiR-432 abundance was highest at the earliest stage of fetal development tested (60 day-old fetus) and decreased throughout development to the adult. Conversely, miR-24 and miR-27 exhibited greatest abundance in proliferating satellite cells and the adult, while abundance of miR-368, miR-376, and miR-423-5p was greatest in the neonate. Conclusion: These data present a complete set of transcriptome profiles to evaluate miR abundance at specific stages of skeletal muscle growth in swine. Identification of these miR provides an initial group of miR that may play a vital role in muscle development and growth.

Changes in postnatal skeletal muscle development induced by alternative immobilization model in female rat

The objective of this study was to adapt a model of hind limb immobilization to newly weaned female rats and to determine the morphology of shortened soleus and plantaris muscles. Female Wistar rats were divided into three groups: control zero (n = 3) and control and free (n = 8), animals aged 21 and 31 days, respectively, submitted to no intervention, and immobilized (n = 25), animals aged 21 days submitted to immobilization for 10 days and sacrificed at 31 days of age. The device used for immobilization had advantages such as easy connection, good fit, and low cost. The immobilized rats showed a reduction in muscle fiber area and in connective tissue. The adaptation of this immobilization model originally used for adult rats was an excellent alternative for newly weaned rats and was also efficient in inducing significant hind limb disuse.

A hypoplastic model of skeletal muscle development displaying reduced foetal myoblast cell numbers, increased oxidative myofibres and improved specific tension capacity

The major component of skeletal muscle is the myofibre. Genetic intervention inducing over-enlargement of myofibres beyond a certain threshold through acellular growth causes a reduction in the specific tension generating capacity of the muscle. However the physiological parameters of a genetic model that harbours reduced skeletal muscle mass have yet to be analysed. Genetic deletion of Meox2 in mice leads to reduced limb muscle size and causes some patterning defects. The loss of Meox2 is not embryonically lethal and a small percentage of animals survive to adulthood making it an excellent model with which to investigate how skeletal muscle responds to reductions in mass. In this study we have performed a detailed analysis of both late foetal and adult muscle development in the absence of Meox2. In the adult, we show that the loss of Meox2 results in smaller limb muscles that harbour reduced numbers of myofibres. However, these fibres are enlarged. These myofibres display a molecular and metabolic fibre type switch towards a more oxidative phenotype that is induced through abnormalities in foetal fibre formation. In spite of these changes, the muscle from Meox2 mutant mice is able to generate increased levels of specific tension compared to that of the wild type.

Regulation of Myosin Heavy Chain Expression during Rat Skeletal Muscle Development In Vitro

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and ∼10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN–nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.

Characterisation of dystrophin during development of human skeletal muscle.

Dystrophin, the 427 x 10(3) Mr product of the Duchenne muscular dystrophy (DMD) gene, was studied in human foetal skeletal muscle from 9 to 26 weeks of gestation. Dystrophin could be detected from at least 9 weeks of gestation at the sarcolemmal membrane of most myotubes, though there was differential staining with antibodies raised to various regions of the protein. Dystrophin immunostaining increased and became more uniform with age and by 26 weeks of gestation there was intense sarcolemmal staining of all myotubes. On a Western blot, a doublet of smaller relative molecular mass than that seen in adult tissue was detected in all foetuses studied. There was a gradual increase in abundance of the upper band from 9 to 26 weeks, and the lower band, although present in low amounts in young foetuses, increased significantly between 20 and 26 weeks of gestation. These data indicate that there are several specific isoforms of dystrophin present in developing skeletal muscle, though the role of these is unknown.

Formoterol treatment downregulates the myostatin system in skeletal muscle of cachectic tumour-bearing rats

Cachexia is a common systemic manifestation. Additionally, myostatin is known to be a negative regulator of skeletal muscle development. The present study aimed to investigate whether formoterol down-regulates the myostatin system in skeletal muscle of tumour-bearing rats. Real-time PCR and Western blotting were used for the analysis. Results showed that rats bearing the Yoshida AH-130 ascites hepatoma, a cachexia-inducing tumour, exhibited marked muscle wasting that affected the mass of the muscles studied. The cachectic animals exhibited a significant increase in the mRNA levels of the myostatin receptor (ActIIB) in gastrocnemius muscles. Notably, the expression of the various forms of follistatin, a protein with the opposite effects to those of myostatin, was significantly reduced as a result of the implantation of the tumour. When the animals were treated with formoterol, a β-agonist with anti-cachectic potential, increases in skeletal muscle weights were observed. The β-agonist significantly increased levels of various follistatin isoforms and significantly decreased the expression levels of the myostatin receptor. In addition, formoterol treatment resulted in a significant decrease of the myostatin protein content of the gastrocnemius muscle. In conclusion, the results presented indicate that certain anabolic actions of formoterol on the skeletal muscle of cachectic animals may be mediated via the myostatin system.

Differentially transcribed genes in skeletal muscle of lambs

The objective of this study was to compare gene transcription profiles in Longissimus dorsi muscle of the following four hair sheep genetic groups, Morada Nova (MO), Brazilian Somali (SO), Santa Inĉs (SI) and 1/2 Dorper×1/2 Morada Nova (F1). These groups all display different postnatal muscle growth. The transcriptomes of the skeletal muscle of the lambs (at 200 days of age) were profiled by using oligonucleotide microarrays and reverse transcription-quantitative real-time PCR (RT-qPCR). The microarray experiment identified 262 transcripts that were differentially expressed when transcription levels were compared between the different breeds. A total of 23 transcripts among which those involved in skeletal muscle development (MyoD1 and IGFBP4), lipogenesis and adipogenesis (C/EBPδ, PPARγ and PGDS) were differentially expressed in at least in one comparison. Clustering analysis showed that there is greater similarity in gene expression between the MO and SI breeds and between F1 and SO genetic groups. The SO breed has the most distinct expression pattern. The RT-qPCR results confirmed the findings from the microarray study. A positive correlation was observed between the expression of MyoD1 and the cold carcass yield. The negative correlations between the weight and yield of cold carcass with the expression of C/EBPδ mean that the selection for adipogenesis could lead to a lower carcass weight. The GLUT3 and PYGL gene transcripts were negatively correlated with fat thickness, but ATP5G1 was positively correlated with this trait. Interestingly, many genes negatively correlated with PUFA were positively correlated with cold carcass yield. In conclusion, the present work demonstrated that there are breed-specific expression patterns in Brazilian hair sheep genetic groups. The differences in gene expression among genetic groups were consistent with their phenotypic differences. The positive correlation of the MyoD1 expression with the cold carcass yield suggests that this gene is important for tissue growth in sheep. The positive correlation of the C/EBPδ expression with PUFA provides an opportunity to select for lipid deposition in meat animals. © 2012 Elsevier B.V.

MicroRNA-499 expression distinctively correlates to target genes sox6 and rod1 profiles to resolve the skeletal muscle phenotype in nile tilapia

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

ELUCIDATING FUNCTIONAL ROLES FOR MYOGENIN IN ADULT SKELETAL MUSCLE METABOLISM, EXERCISE CAPACITY, AND REGENERATION

The four basic helix-loop-helix myogenic transcription factors, myogenin, Myf5, MRF4, and MyoD are critical for embryonic skeletal muscle development. Myogenin is necessary for the terminal differentiation of myoblasts into myofibers during embryogenesis, but little is known about the roles played by myogenin in adult skeletal muscle function and metabolism. Furthermore, while metabolism is a well-studied physiological process, how it is regulated at the transcriptional level remains poorly understood. In this study, my aim was to determine the function of myogenin in adult skeletal muscle metabolism, exercise capacity, and regeneration. To investigate this, I utilized a mouse strain harboring the Myogflox allele and a Cre recombinase transgene, enabling the efficient deletion of myogenin in the adult mouse. Myogflox/flox mice were stressed physically through involuntary treadmill running and by breeding them with a strain harboring the Duchenne’s muscular dystrophy (DMDmdx) allele. Surprisingly, Myog-deleted animals exhibited an enhanced capacity for exercise, running farther and faster than their wild-type counterparts. Increased lactate production and utilization of glucose as a fuel source indicated that Myog-deleted animals exhibited an increased glycolytic flux. Hypoglycemic Myog-deleted mice no longer possessed the ability to outrun their wild-type counterparts, implying the ability of these animals to further deplete their glucose reserves confers their enhanced exercise capacity. Moreover, Myog-deleted mice exhibited an enhanced response to long-term exercise training. The mice developed a greater proportion of type 1 oxidative muscle fibers, and displayed increased levels of succinate dehydrogenase activity, indicative of increased oxidative metabolism. Mdx:Myog-deleted mice exhibited a similar phenotype, outperforming their mdx counterparts, although lagging behind wild-type animals. The morphology of muscle tissue from mdx:Myog-deleted mice appears to mimic that of mdx animals, indicating that myogenin is dispensable for adult skeletal muscle regeneration. Through global gene expression profiling and quantitative (q)RT-PCR, I identified a unique set of putative myogenin-dependent genes involved in regulating metabolic processes. These data suggest myogenin’s functions during adulthood are distinctly different than those during embryogenesis, and myogenin acts as a high-level transcription factor regulating metabolic activity in adult skeletal muscle.

Combinatorial control of muscle development by basic helix-loop-helix and MADS-box transcription factors.

Members of the MyoD family of muscle-specific basic helix-loop-helix (bHLH) proteins function within a genetic pathway to control skeletal muscle development. Mutational analyses of these factors suggested that their DNA binding domains mediated interaction with a coregulator required for activation of muscle-specific transcription. Members of the myocyte enhancer binding factor 2 (MEF2) family of MADS-box proteins are expressed at high levels in muscle and neural cells and at lower levels in several other cell types. MEF2 factors are unable to activate muscle gene expression alone, but they potentiate the transcriptional activity of myogenic bHLH proteins. This potentiation appears to be mediated by direct interactions between the DNA binding domains of these different types of transcription factors. Biochemical and genetic evidence suggests that MEF2 factors are the coregulators for myogenic bHLH proteins. The presence of MEF2 and cell-specific bHLH proteins in other cell types raises the possibility that these proteins may also cooperate to regulate other programs of cell-specific gene expression. We present a model to account for such cooperative interactions.

Aberrant repair and fibrosis development in skeletal muscle

The repair process of damaged tissue involves the coordinated activities of several cell types in response to local and systemic signals. Following acute tissue injury, infiltrating inflammatory cells and resident stem cells orchestrate their activities to restore tissue homeostasis. However, during chronic tissue damage, such as in muscular dystrophies, the inflammatory-cell infiltration and fibroblast activation persists, while the reparative capacity of stem cells (satellite cells) is attenuated. Abnormal dystrophic muscle repair and its end stage, fibrosis, represent the final common pathway of virtually all chronic neurodegenerative muscular diseases. As our understanding of the pathogenesis of muscle fibrosis has progressed, it has become evident that the muscle provides a useful model for the regulation of tissue repair by the local microenvironment, showing interplay among muscle-specific stem cells, inflammatory cells, fibroblasts and extracellular matrix components of the mammalian wound-healing response. This article reviews the emerging findings of the mechanisms that underlie normal versus aberrant muscle-tissue repair.

Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

The present study analyzes the ectopic development of the rat skeletal muscle originated from transplanted satellite cells. Satellite cells (10(6) cells) obtained from hindlimb muscles of newborn female 2BAW Wistar rats were injected subcutaneously into the dorsal area of adult male rats. After 3, 7, and 14 days, the transplanted tissues (N = 4-5) were processed for histochemical analysis of peripheral nerves, inactive X-chromosome and acetylcholinesterase. Nicotinic acetylcholine receptors (nAChRs) were also labeled with tetramethylrhodamine-labeled alpha-bungarotoxin. The development of ectopic muscles was successful in 86% of the implantation sites. By day 3, the transplanted cells were organized as multinucleated fibers containing multiple clusters of nAChRs (N = 2-4), resembling those from non-innervated cultured skeletal muscle fibers. After 7 days, the transplanted cells appeared as a highly vascularized tissue formed by bundles of fibers containing peripheral nuclei. The presence of X chromatin body indicated that subcutaneously developed fibers originated from female donor satellite cells. Differently from the extensor digitorum longus muscle of adult male rat (87.9 ± 1.0 µm; N = 213), the diameter of ectopic fibers (59.1 µm; N = 213) did not obey a Gaussian distribution and had a higher coefficient of variation. After 7 and 14 days, the organization of the nAChR clusters was similar to that of clusters from adult innervated extensor digitorum longus muscle. These findings indicate the histocompatibility of rats from 2BAW colony and that satellite cells transplanted into the subcutaneous space of adult animals are able to develop and fuse to form differentiated skeletal muscle fibers.

Effects of Low-Level Laser Therapy (LLLT) in the Development of Exercise-Induced Skeletal Muscle Fatigue and Changes in Biochemical Markers Related to Postexercise Recovery

STUDY DESIGN: Randomized crossover double-blinded placebo-controlled trial. OBJECTIVE: To investigate if low-level laser therapy (LLLT) can affect biceps muscle performance, fatigue development, and biochemical markers of postexercise recovery. BACKGROUND: Cell and animal studies have suggested that LLLT can reduce oxidative stress and inflammatory responses in muscle tissue. But it remains uncertain whether these findings can translate into humans in sport and exercise situations. METHODS: Nine healthy male volleyball players participated in the study. They received either active LLLT (cluster probe with 5 laser diodes; A = 810 nm; 200 mW power output; 30 seconds of irradiation, applied in 2 locations over the biceps of the nondominant arm; 60 J of total energy) or placebo LLLT using an identical cluster probe. The intervention or placebo were applied 3 minutes before the performance of exercise. All subjects performed voluntary elbow flexion repetitions with a workload of 75% of their maximal voluntary contraction force until exhaustion. RESULTS: Active LLLT increased the number of repetitions by 14.5% (mean +/- SD, 39.6 +/- 4.3 versus 34.6 +/- 5.6; P = .037) and the elapsed time before exhaustion by 8.0% (P = .034), when compared to the placebo treatment. The biochemical markers also indicated that recovery may be positively affected by LLLT, as indicated by postexercise blood lactate levels (P<.01), creatine kinase activity (P = .017), and C-reactive protein levels (P = .047), showing a faster recovery with LLLT application prior to the exercise. CONCLUSION: We conclude that pre-exercise irradiation of the biceps with an LLLT dose of 6 J per application location, applied in 2 locations, increased endurance for repeated elbow flexion against resistance and decreased postexercise levels of blood lactate, creatine kinase, and C-reactive protein. LEVEL OF EVIDENCE: Performance enhancement, level 1b. J Orthop Sports Phys Ther 2010;40(8):524-532. doi:10.2519/jospt.2010.3294

Development of a continuum mechanics model of passive skeletal muscle

Skeletal muscle force evaluation is difficult to implement in a clinical setting. Muscle force is typically assessed through either manual muscle testing, isokinetic/isometric dynamometry, or electromyography (EMG). Manual muscle testing is a subjective evaluation of a patient’s ability to move voluntarily against gravity and to resist force applied by an examiner. Muscle testing using dynamometers adds accuracy by quantifying functional mechanical output of a limb. However, like manual muscle testing, dynamometry only provides estimates of the joint moment. EMG quantifies neuromuscular activation signals of individual muscles, and is used to infer muscle function. Despite the abundance of work performed to determine the degree to which EMG signals and muscle forces are related, the basic problem remains that EMG cannot provide a quantitative measurement of muscle force. Intramuscular pressure (IMP), the pressure applied by muscle fibers on interstitial fluid, has been considered as a correlate for muscle force. Numerous studies have shown that an approximately linear relationship exists between IMP and muscle force. A microsensor has recently been developed that is accurate, biocompatible, and appropriately sized for clinical use. While muscle force and pressure have been shown to be correlates, IMP has been shown to be non-uniform within the muscle. As it would not be practicable to experimentally evaluate how IMP is distributed, computational modeling may provide the means to fully evaluate IMP generation in muscles of various shapes and operating conditions. The work presented in this dissertation focuses on the development and validation of computational models of passive skeletal muscle and the evaluation of their performance for prediction of IMP. A transversly isotropic, hyperelastic, and nearly incompressible model will be evaluated along with a poroelastic model.

Scatter factor/hepatocyte growth factor as a regulator of skeletal muscle and neural crest development.

Factors that regulate cellular migration during embryonic development are essential for tissue and organ morphogenesis. Scatter factor/hepatocyte growth factor (SF/HGF) can stimulate motogenic and morphogenetic activities in cultured epithelial cells expressing the Met tyrosine kinase receptor and is essential for development; however, the precise physiological role of SF/HGF is incompletely understood. Here we provide functional evidence that inappropriate expression of SF/HGF in transgenic mice influences the development of two distinct migratory cell lineages, resulting in ectopic skeletal muscle formation and melanosis in the central nervous system, and patterned hyperpigmentation of the skin. Committed TRP-2 positive melanoblasts were found to be situated aberrantly within defined regions of the transgenic embryo, including the neural tube, which overproduced SF/RGF. Our data strongly suggest that SF/HGF possesses physiologically relevant scatter activity, and functions as a true morphogenetic factor by regulating migration and/or differentiation of select populations of premyogenic and neural crest cells during normal mammalian embryogenesis.