Time course of myogenic and metabolic gene expression in response to acute exercise in human skeletal muscle.

The aim of this study was to examine the time course activation of select myogenic (MRF4, Myf5, MyoD, myogenin) and metabolic (CD36, CPT1, HKII, and PDK4) genes after an acute bout of resistance (RE) or run (Run) exercise. Six RE subjects [25 +/- 4 yr (mean +/- SD), 74 +/- 14 kg, 1.71 +/- 0.11 m] and six Run subjects (25 +/- 4 yr, 72 +/- 5 kg, 1.81 +/- 0.07 m, 63 +/- 8 ml.kg(-1).min(-1)) were studied. Eight muscle biopsies were taken from the vastus lateralis (RE) and gastrocnemius (Run) before, immediately after, and 1, 2, 4, 8, 12 and 24 h after exercise. RE increased mRNA of MRF4 (3.7- to 4.5-fold 2-4 h post), MyoD (5.8-fold 8 h post), myogenin (2.6- and 3.5-fold 8-12 h post), HKII (3.6- to 10.5-fold 2-12 h post), and PDK4 (14- to 26-fold 2-8 h post). There were no differences in Myf5, CD36, and CPT1 mRNA levels 0-24 h post-RE. Run increased mRNA of MyoD (5.0- to 8.0-fold), HKII (12- to 16-fold), and PDK4 (32- to 52-fold) at 8-12 h postexercise. There were no differences in MRF4, Myf5, myogenin, CD36 and CPT1 mRNA levels 0-24 h post-Run. These data indicate a myogenic and metabolic gene induction with RE and Run exercise. The timing of the gene induction is variable and generally peaks 4-8 h postexercise with all gene expression not significantly different from the preexercise levels by 24 h postexercise. These data provide basic information for the timing of human muscle biopsy samples for gene-expression studies involving exercise.     

Expression of myogenic factors in denervated chicken breast muscle: isolation of the chicken Myf5 gene.

In this study, we have isolated and characterized the chicken Myf5 gene, and cDNA clones encoding chicken MyoD1 and myogenin. The chicken Myf5 and MRF4 genes are tandemly located on a single genomic DNA fragment, and the chicken Myf5 gene is organized into at least three exons. Using genomic and cDNA probes, we further analyzed the mRNA levels of four myogenic factors during chicken breast muscle development. This analysis revealed that myogenin expression is restricted to in ovo stages in breast muscle, and is not detectable in neonatal and adult stages. On the other hand, Myf5 expression is detectable until day 7 post-hatching, and is not found in adult muscle, whereas high levels of MyoD1 and MRF4 are detectable at all stages. To further understand the roles of innervation on muscle maturation, we analyzed the expression of the four myogenic factors in denervated adult breast muscle. We found that MyoD1, myogenin, and MRF4 are induced at high levels in denervated muscle, whereas no change occurs in the level of Myf5. These studies suggest that innervation controls the relative abundance and type of myogenic factors that are expressed in adult muscle, and that when nerve control is removed, the muscle reverts to a neonatal phenotype, with the enhanced expression of three myogenic factors (MyoD1, myogenin, and MRF4).     

Myostatin and MyoD family expression in skeletal muscle of IGF-1 knockout mice

Insulin-like growth factor-1 (IGF-1) is a positive regulator in proliferation and differentiation of skeletal muscle cells, while myostatin (MSTN) is a member of transforming growth factor beta superfamily that acts as a negative regulator of skeletal muscle mass. The present study was performed to detail whether a correlation exists between MSTN and IGF-1 in skeletal muscle of IGF-1 knockout mice (IGF-1(-/-)) and their wild type (WT; i.e., IGF-1(+/+)) littermates. The body weight of IGF-1(-/-) animals was 32% that of WT littermates. The fiber cross-sectional areas (CSA) and number of fibers in M. rectus femoris of IGF-1(-/-) animals were 49 and 59% those of WT animals, respectively. Thus, muscle hypoplasia of IGF-1(-/-) undoubtedly was confirmed. Myostatin mRNA levels and protein levels were similar between M. gastrocnemius of IGF-1(-/-) and WT animals. Myostatin immunoreactivity was similarly localized in muscle fibers of both IGF-1(-/-) and WT M. rectus femoris. The mRNA levels of MyoD family (Myf5, MyoD, MRF4, myogenin) were differentially expressed in IGF-1(-/-)M. gastrocnemius, in which the mRNA expression of MRF4 and myogenin was significantly lower, whereas there were no changes in the mRNA expression of Myf5 and MyoD. These findings first describe that myostatin expression is not influenced by intrinsic failure of IGF-1, although MRF4 and myogenin are downregulated.     

The expression of myogenic regulatory factors and muscle growth factors in the masticatory muscles of dystrophin-deficient (MDX) mice

The activities of myogenic regulatory factors (MRF) and muscle growth factors increase in muscle that is undergoing regeneration, and may correspond to some specific changes. Little is known about the role of MRFs in masticatory muscles in mdx mice (the model of Duchenne muscular dystrophy) and particularly about their mRNA expression during the process of muscle regeneration. Using Taqman RT-PCR, we examined the mRNA expression of the MRFs myogenin and MyoD1 (myogenic differentiation 1), and of the muscle growth factors myostatin, IGF1 (insulin-like growth factor) and MGF (mechanogrowth factor) in the masseter, temporal and tongue masticatory muscles of mdx mice (n = 6 to 10 per group). The myogenin mRNA expression in the mdx masseter and temporal muscle was found to have increased (P < 0.05), whereas the myostatin mRNA expressions in the mdx masseter (P < 0.005) and tongue (P < 0.05) were found to have diminished compared to those for the controls. The IGF and MGF mRNA amounts in the mdx mice remained unchanged. Inside the mdx animal group, gender-related differences in the mRNA expressions were also found. A higher mRNA expression of myogenin and MyoD1 in the mdx massterer and temporal muscles was found in females in comparison to males, and the level of myostatin was higher in the masseter and tongue muscle (P < 0.001 for all comparisons). Similar gender-related differences were also found within the control groups. This study reveals the intermuscular differences in the mRNA expression pattern of myogenin and myostatin in mdx mice. The existence of these differences implies that dystrophinopathy affects the skeletal muscles differentially. The finding of gender-related differences in the mRNA expression of the examined factors may indicate the importance of hormonal influences on muscle regeneration.     

Conditional conversion of ES cells to skeletal muscle by an exogenous MyoD1 gene.

A variety of differentiated cell types can be converted to skeletal muscle cells following transfection with the myogenic regulatory gene MyoD1. To determine whether multipotent embryonic stem (ES) cells respond similarly, cultures of two ES cell lines were electroporated with a MyoD1 cDNA driven by the beta-actin promoter. All transfected clones, carrying a single copy of the exogenous gene, expressed high levels of MyoD1 mRNA. Surprisingly, although maintained in mitogen-rich medium, this ectopic expression was associated with a transactivation of the endogenous myogenin and myosin light chain 2 gene but not the endogenous MyoD1, MRF4, Myf5, the skeletal muscle actin, or the myosin heavy chain genes. Preferential myogenesis and the appearance of contracting skeletal muscle fibers were observed only when the transfected cells were allowed to differentiate in vitro, via embryoid bodies, in low-mitogen-containing medium. Myogenesis was associated with the activation of MRF4 and Myf5 genes and resulted in a significant increase in the level of myogenin mRNA. Not all cells were converted to skeletal muscle cells, indicating that only a subset of stem cells can respond to MyoD1. Moreover, the continued expression of the introduced gene was not required for myogenesis. These results show that ES cells can respond to MyoD1, but environmental factors control the expression of its myogenic differentiation function, that MyoD1 functions in ES cells even under environmental conditions that favor differentiation is not dominant (incomplete penetrance), that MyoD1 expression is required for the establishment of the myogenic program but not for its maintenance, and that the exogenous MyoD1 gene can trans-activate the endogenous myogenin and MLC2 genes in undifferentiated ES cells.     

Involvement of myogenic regulator factors during fusion in the cell line C2C12

The myogenic factors, MyoD, myogenin, Myf5 and MRF4, can activate skeletal muscle differentiation when overexpressed in non-muscular cells. Gene targeting experiments have provided much insight into the in vivo functions of MRF and have defined two functional groups of MRFs. MyoD and Myf5 may be necessary for myoblast determination while myogenin and MRF4 may be required later during differentiation. However, the specific role of these myogenic factors has not been clearly defined during one important stage of myogenesis: the fusion of myoblasts. Using cultured C2C12 mouse muscular cells, the time-course of these proteins was analyzed and a distinct expression pattern in fusing cells was revealed. In an attempt to clarify the role of each of these regulators during myoblast fusion, an antisense strategy using oligonucleotides with phosphorothioate backbone modification was adoped. The results showed that the inhibition of myogenin and Myf5 activity is capable of significantly preventing fusion. Furthermore, the inhibition of MyoD can wholly arrest the engaged fusion process in spite of high endogenous expression of both myogenin and Myf5. Consequently, each MRF seems to have, at this defined step of myogenesis, a specific set of functions that can not be substituted for by the others and therefore may regulate a distinct subset of muscle-specific genes at the onset of fusion.     

Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products.

The development of muscle cells involves the action of myogenic determination factors. In this report, we show that human skeletal muscle tissue contains, besides the previously described Myf-5, two additional factors Myf-3 and Myf-4 which represent the human homologues of the rodent proteins MyoD1 and myogenin. The genes encoding Myf-3, Myf-4 and Myf-5 are located on human chromosomes 11, 1, and 12 respectively. Constitutive expression of a single factor is sufficient to convert mouse C3H 10T1/2 fibroblasts to phenotypically normal muscle cells. The myogenic conversion of 10T1/2 fibroblasts results in the activation of the endogenous MyoD1 and Myf-4 (myogenin) genes. This observation suggests that the expression of Myf proteins leads to positive autoregulation of the members of the Myf gene family. Individual myogenic colonies derived from MCA C115 cells (10T1/2 fibroblast transformed by methylcholanthrene) express various levels of endogenous MyoD1 mRNA ranging from nearly zero to high levels. The Myf-5 gene was generally not activated in 10T1/2 derived myogenic cell lines but was expressed in some MCA myoblasts. In primary human muscle cells Myf-3 and Myf-4 mRNA but very little Myf-5 mRNA is expressed. In mouse C2 and P2 muscle cell lines MyoD1 is abundantly synthesized together with myogenin. In contrast, the rat muscle lines L8 and L6 and the mouse BC3H1 cells express primarily myogenin and low levels of Myf-5 but no MyoD1. Myf-4 (myogenin) mRNA is present in all muscle cell lines at the onset of differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suppression of myostatin with vector-based RNA interference causes a double-muscle effect in transgenic zebrafish

Myostatin belongs to the transforming growth factor (TGF)-β superfamily and is a potent negative regulator of skeletal muscle development and growth. We utilized microinjection of an antisense RNA-expressing vector to establish a hereditarily stable myostatin gene knockdown zebrafish strain with a double-muscle phenotype. Real-time PCR and immunostaining revealed that the myostatin messenger (m)RNA and protein levels in homozygous transgenic zebrafish were 33% and 26% those of the non-transgenic controls, respectively. Also, the mRNA levels of myogenic regulatory factor markers such as MyoD, myogenin, Mrf4, and Myf5 were dramatically elevated in myostatin-suppressed transgenic fish compared to the non-transgenic controls. Although there was no significant difference in body length, homozygous transgenic zebrafish were 45% heavier than non-transgenic controls. Histochemical analysis showed that the cross-sectional area of the muscle fiber of homozygous transgenic fish was twice as large as that of non-transgenic controls. This is the first model zebrafish with a hereditarily stable myostatin-suppressed genotype and a double-muscle phenotype.