Myogenic Phases Research Articles

The overlap between regeneration and fibrosis in injured skeletal muscle is regulated by phosphatidylinositol 3-kinase/Akt signaling pathway - A bioinformatic analysis based on lncRNA microarray

Injured skeletal muscle would go through a sequence of the pathological phases of degeneration, myogenesis and fibrosis. Growing evidence indicated that fibrotic and myogenic phases might overlap within the injured skeletal muscle in the early time after injury. However, the mechanism underlying this overlapping remains unclear. Here, we performed an lncRNA microarray to identify the activated pathways in mice muscle seven days after contusion. KEGG analysis indicated that phosphatidylinositol 3-kinase/Akt (PI3K/Akt) signaling cascade was predicted to be activated by lncRNAs. The top genes targeted by lncRNAs in PI3K/Akt signaling were subunits of laminin, collagen 5, and collagen 6, which participated in either myogenic or fibrotic process. Reverse transcriptase–polymerase chain reaction analysis and immunohistochemical stain further confirmed the prediction in silico. These results suggested that the overlap might be related to an activated PI3K/Akt pathway by lncRNA regulation.

MyoD Overexpressed Equine Adipose-Derived Stem Cells Enhanced Myogenic Differentiation Potential.

Mesenchymal stem cells could potentially be used in the clinical treatment of muscle disorders and muscle regeneration. Adipose-derived stem cells (ADSCs) can be easily isolated from adipose tissue, as opposed to stem cells of other tissues. We believe that cell therapy using ADSCs could be applied to muscle disorders in horses and other species. We sought to improve the myogenic differentiation potential of equine ADSCs (eqADSCs) using a MyoD lentiviral vector. MyoD lentiviruses were transduced into eqADSCs and selected using puromycin. Cells were cultured in differentiation media containing 5% horse serum, and after 5 days the MyoD-transduced cells differentiated into myogenic cells (MyoD-eqADSCs). Using green fluorescent protein (GFP), MyoD-eqADSCs were purified and transplanted into the tibialis anterior muscles of mice after they were injured with the myotoxin notexin. The mice were sacrificed to examine any regeneration in the tibialis anterior muscle 4 weeks after the MyoD-eqADSCs were injected. The MyoD-eqADSCs cultured in growth media expressed murine and equine MyoD; however, they did not express late differentiation markers such as myogenin (MYOG). When cells were grown in differentiation media, the expression of MYOG was clearly observed. According to our reverse transcription polymerase chain reaction and immunocytochemistry results, MyoD-eqADSCs expressed terminal myogenic phase genes, such as those encoding dystrophin, myosin heavy chain, and troponin I. The MyoD-eqADSCs fused to each other, and the formation of myotube-like cells from myoblasts in differentiation media occurred between days 5 and 14 postplating. In mice, we observed GFP-positive myofibers, which had differentiated from the injected MyoD-eqADSCs. Our approaches improved the myogenic differentiation of eqADSCs through the forced expression of murine MyoD. Our findings suggest that limitations in the treatment of equine muscle disorders could be overcome using ADSCs.

Effective myotube formation in human adipose tissue-derived stem cells expressing dystrophin and myosin heavy chain by cellular fusion with mouse C2C12 myoblasts

Stem cell therapy for muscular dystrophies requires stem cells that are able to participate in the formation of new muscle fibers. However, the differentiation steps that are the most critical for this process are not clear. We investigated the myogenic phases of human adipose tissue-derived stem cells (hASCs) step by step and the capability of myotube formation according to the differentiation phase by cellular fusion with mouse myoblast C2C12 cells. In hASCs treated with 5-azacytidine and fibroblast growth factor-2 (FGF-2) for 1 day, the early differentiation step to express MyoD and myogenin was induced by FGF-2 treatment for 6 days. Dystrophin and myosin heavy chain (MyHC) expression was induced by hASC conditioned medium in the late differentiation step. Myotubes were observed only in hASCs undergoing the late differentiation step by cellular fusion with C2C12 cells. In contrast, hASCs that were normal or in the early stage were not involved in myotube formation. Our results indicate that stem cells expressing dystrophin and MyHC are more suitable for myotube formation by co-culture with myoblasts than normal or early differentiated stem cells expressing MyoD and myogenin.

Altered Primary and Secondary Myogenesis in the Myostatin-Null Mouse

Skeletal muscle fiber generation occurs principally in two myogenic phases: (1) Primary (embryonic) myogenesis when myoblasts proliferate and fuse to form primary myotubes and (2) secondary (fetal) myogenesis when successive waves of myoblasts fuse along the surface of the primary myotubes, giving rise to a population of smaller and more numerous secondary myotubes. This sequence of events determines fiber number and is completed at or soon after birth in most muscles of the mouse. The adult myostatin null mouse (MSTN(-/-)) displays both an increase in fiber number and size relative to wild type (MSTN(+/+)), suggesting a developmental origin for the hypermuscular phenotype. The focus of the present study was to determine at which point during myogenesis do MSTN(-/-) animals diverge from MSTN(+/+). To achieve this, we focused on the extensor digitorum longus (EDL) muscle and evaluated primary myotube number at embryonic day (E) 13.0 and E14.5 and secondary to primary myotube ratios at E18.5. We show that primary myotube number and size were significantly increased in the MSTN(-/-) mice by E14.5 and the secondary to primary myotube ratio increased at E18.5. This increase in the rate of fiber formation resulted in MSTN(-/-) mice harboring 87% of their final adult fiber number at E18.5, compared to only 73% in MSTN(+/+). An accelerated myogenic program in the MSTN(-/-) mice was further confirmed by our finding of an initial expansion in the myogenic stem cell (identified through Pax7 expression) and myoblast (identified through myogenin expression) cell pools at E14.5 in the EDL muscle of these animals that was, however, followed by a reduction of both populations of cells at E18.5 relative to MSTN(+/+). Overall these data suggest that the genetic loss of myostatin accelerates the developmental myogenic program of primary and secondary skeletal myogenesis.

Fortalecimiento muscular, nivel de fuerza muscular y autonomía funcional en una población de mujeres mayores

Introduction This study aimed to evaluate the effects of muscular strengthening on the level of muscular strength in the neurogenic and myogenic phases and functional autonomy in a population of healthy, sedentary, elderly women. Material and methods The sample was composed of 40 women, randomly divided into an experimental group (n=20; 65.62±5.36 years) and a control group (n=20; 71.45±5.72). A protocol of one repetition maximum (1 RM) was employed to evaluate muscular strength and the battery of tests included in the protocol of the Latin American Development Group for Maturity was used to evaluate functional autonomy. For the statistical analysis, the following tests were used: Kruskal-Wallis (experimental group in relation to three moments) followed by Dunn's multiple comparison test; Student's t-test for dependent samples (control group in relation to two moments) and the Mann-Whitney and Student's t-test for independent samples for Δ% (inter-group comparison). Results The results in the experimental group showed a significant increase in the myogenic phase in comparison with the pre-test (p-values: knee extension =0.0001; right knee flexion and left knee flexion=0.0001; straight supine=0.0001; triceps curl=0.0001. Functional autonomy tests showed significant improvements in the experimental group: neurogenic phase (p-values: general autonomy index=0.0089; walking 10 meters=0.0106; standing from a sitting position =0.0005; standing from a ventral decubitus position =0.0061; standing from a sitting position and walking around the house =0.0072; putting on and taking off a shirt =0.0104) and the myogenic phase (p-values: general autonomy index=0.0001; walking 10 meters=0.0005; standing from a sitting position =0.0000; standing from a ventral decubitus position =0.004; standing from a sitting position and walking around the house =0.0059; putting on and taking off a shirt =0.0003). Conclusions Thus, strength training only showed statistically significant differences in the myogenic phase; statistically significant reductions were also found in the time needed to perform functional autonomy tests in the neurogenic phase.

Renal blood flow and dynamic autoregulation in conscious mice

Autoregulation of renal blood flow (RBF) occurs via myogenic and tubuloglomerular feedback (TGF) mechanisms that are engaged by pressure changes within preglomerular arteries and by tubular flow and content, respectively. Our understanding of autoregulatory function in the kidney largely stems from experiments in anesthetized animals where renal perfusion pressure is precisely controlled. However, normally occurring variations in blood pressure are sufficient to engage both myogenic and TGF mechanisms, making the assessment of autoregulatory function in conscious animals of significant value. To our knowledge, no studies have evaluated the dynamics of RBF in conscious mice. Therefore, we used spectral analysis of blood pressure and RBF and identified dynamic operational characteristics of the myogenic and TGF mechanisms in conscious, freely moving mice instrumented with ultrasound flow probes and arterial catheters. The myogenic response generates a distinct resonance peak in transfer gain at 0.31 +/- 0.01 Hz. Myogenic-dependent attenuation of RBF oscillations, indicative of active autoregulation, is apparent as a trough in gain below 0.3 Hz (-6.5 +/- 1.3 dB) and a strong positive phase peak (93 +/- 9 deg), which are abolished by amlodipine infusion. Operation of TGF produces a local maximum in gain at 0.05 +/- 0.01 Hz and a positive phase peak (62.3 +/- 12.3 deg), both of which are eliminated by infusion of furosemide. Administration of amlodipine eliminated both myogenic and TGF signature peaks, whereas furosemide shifted the myogenic phase peak to a slower operational frequency. These data indicate that myogenic and TGF dynamics may be used to investigate the effectiveness of renal autoregulatory mechanisms in conscious mice.

MyoD and Myogenin expression during myogenic phases in brown trout: A precocious onset of mosaic hyperplasia is a prerequisite for fast somatic growth

Muscle cell recruitment (hyperplasia) during myogenesis in the vertebrate embryo is known to occur in three consecutive phases. In teleost fish (including zebrafish), however, information on myogenic precursor cell activation is largely fragmentary, and comprehensive characterization of the myogenic phases has only been fully undertaken in a single slow-growing cyprinid species by examination of MEF2D expression. Here, we use molecular techniques to provide a comprehensive characterization of MyoD and Myogenin expression during myogenic cell activation in embryos and larvae of brown trout, a fast-growing salmonid with exceptionally large embryos. Results confirm the three-phase pattern, but also demonstrate that the second and third phases begin simultaneously and progress vigorously, which is different from the previously described consecutive activation of these phases. Furthermore, we suggest that Pax7 is expressed in myogenic progenitor cells that account for second- and third-phase myogenesis. These findings are discussed in relation to teleost myotome development and to teleost growth strategies.

Glycosphingolipid biosynthesis during myogenesis of rat L6 cells in vitro.

Glycosphingolipid biosynthesis was examined using [3H]-galactose as a precursor as rat L6 myoblasts fused to form multinucleated myotubes. Incorporation of label into neutral glycolipids decreased steadily as the population of myotubes increased, so that final biosynthesis was one-half that observed with myoblasts (p less than 0.02). Conversely, ganglioside biosynthesis doubled during myoblast confluency (p less than 0.02) and then decreased as myotubes formed. Qualitatively, L6 cells synthesized large amounts of ganglioside GM3 during all myogenic phases. The major neutral glycosphingolipid products were lactosylceramide and paragloboside (nLcOse4Cer). Few changes in TLC autoradiographic patterns were noted during differentiation, with the exception of a slight decrease in ganglioside GM1. The results indicate that the biosynthesis of glycosphingolipids is tightly regulated during myogenesis in vitro and suggest a role for membrane gangliosides in muscle cell differentiation.

Molecular forms of acetylcholinesterase in developing Torpedo embryos

We have studied the evolution of acetylcholinesterase molecular forms during the embryonic development of Torpedo marmorata, in the electric organs and in the electric lobes of the central nervous system. In the early stages of development (35 mm embryos, ‘myogenic phase’ of electric organ development), globular forms of acetylcholinesterase (G 4 and G 2) are abundant in both tissues and the collagen-tailed form A 12 is already present. In the electric organs, this form accumulates rapidly after the 55–60 mm stage (‘electrogenic phase’), when synapse formation first commences. Although the molecular characteristics of the collagen-tailed forms, and particularly their aggregation properties, do not appear to change during development, their solubilization requires higher concentrations of MgCl 2, as the electrocytes mature, suggesting that they become more tightly integrated in a better organized basal lamina. The smaller collagen-tailed form A 8 shows a transient increase which coincides approximately with the maximal accumulation of A 12, suggesting that it is an intermediate in its synthesis. The accumulation of the hydrophobic G 2, which eventually becomes predominent in the adult electric organs, lags behind that of A 12. The functional significance of this important fraction of acetylcholinesterase is therefore not that of a pool of precursor for the synthesis of A 12. In the electric lobes, the tetrameric form (G 4) is abundant during development, as well as G 2 and G 1 at certain stages, but the A 12 form is predominant in the adult.

Is EmbryonicLimulusHeart Really Myogenic?

Although the neurogenic nature of the heartbeat in adult Limulus has been well studied and is undisputed, we contest the reports that the embryonic heartbeat is myogenic. This notion, based on histological, calorimetric, and drug studies, is challenged by evidence from transmission electron microscopy and intracellular recording. The first, infrequent heartbeats occur at the time of the third embryonic molt when only the anterior portion of the heart tube is formed and functional. Contractions extend further caudad concomitant with lumen formation in the rear heart segments. All lumen-containing heart sections that we have examined, from the earliest on, have revealed neural elements in a bundle at the dorsal midline of the heart. Axons 1/m or less in diameter are prevalent: vesicle-filled terminal-like areas adjacent to muscle cells are often present as well, even in the youngest beating hearts. Myocardial cells show excitatory postsynaptic potentials as soon as heartbeat has begun, but they often fail to summate in the earlier stages so that contractions are few. Resting potentials remain at −65 to −70 mV from the onset of heartbeat until well after the larva has hatched, but heartbeat frequency, regularity, depolarization height (never overshooting) and duration all increase as embryos get older, probably as innervation of muscle fibers increases and coordination between pacemaker and follower neurons improves. We have found no evidence that embryonic Limulus heart passes through a myogenic phase and believe that it is neurally driven from the beginning.

The developmental morphology of Torpedo marmorata: electric organ--myogenic phase.

The early development of the electric organ of Torpedo marmorata has been examined by light and electron microscopy to the 40-mm stage of embryo growth. The myogenic nature of this tissue is confirmed ultrastructurally by the presence of myoblasts and myotubes both containing myofibrils cross striated with Z,A and I bands. Fusion between these cells is also found taking place. A scheme is presented to explain the development of the overall structural plan of the organ and specifically the formation of the future electrocyte columns. AT 40 mm, a series of morphological transformations signals the onset of a divergent developmental pattern ultimately leading to the establishment of mature electrocyte columns. These features include rounding up of myotubes, dissolution of myofibrils and the appearance of intermediate size filaments (11 nm) and perhaps a non-muscular actin (5.5 nm). This early myogenic phase of development occurs in the absence of any specific nervous contact even though electromotor nerves are always in close proximity.