Gene and protein expressions of vimentin and desmin during embryonic development of the mylohyoid muscle.

Meckel's cartilage is known to be involved in formation of the prenatal mandible. However, the relationship between Meckel's cartilage and the embryonic mylohyoid muscle during growth and development has been investigated only rarely. This study examined the expression of intermediate filaments in Meckel's cartilage and the embryonic mylohyoid muscle in fetal mice during morphological development. Specimens of E12-16 ICR mice sectioned in the frontal direction were subjected to immunohistochemistry for vimentin and desmin. Hematoxylin and eosin sections showed that the immature mylohyoid muscle began to grow along Meckel's cartilage during fetal development. Weak vimentin expression was detected in the mylohyoid muscle and surrounding tissues at E12. Desmin expression was detected specifically in the mylohyoid, and strong expression was evident after E13, and increased with age. It was inferred that the mylohyoid muscle is one the tissues developing from Meckel's cartilage, the latter exerting a continuous influence on the growth of the former. In the early stage, the surrounding mesenchymal tissues expressing vimentin formed a scaffold for the developing mylohyoid muscle. Muscle attachment at E13 showed steady desmin expression, which continued until maturity. This study suggested the possibility that Meckel's cartilage has an influence not only on the mandibular bone, but also on the development of the mylohyoid muscle attached to the mandibular bone. Furthermore, it revealed a stage of the developmental process of the mylohyoid muscle in which the expression of vimentin, which is a common protein in the surrounding tissue such as muscle and bone, induces the morphological formation of the mylohyoid muscle, cooperating with the surrounding structures.

Fibroblasts have plasticity and potential utility for cell therapy.

Fibroblasts exist in the interstices of various organs as a component of connective tissue and are one of several types of somatic cells that have been well established in culture. They have been reported to undergo myogenic conversion and to induce the expression of genes associated with pluripotency. However, their own plasticity with regard to direct differentiation has scarcely been described. Here, we show that human fibroblasts are able to differentiate directly to all three germ layer derivatives. The results indicate that human dermal fibroblasts have more plasticity than has been generally thought and that fibroblasts have potential utility as a source for cell therapy.

A comparative study of myostatin, follistatin and decorin expression in muscle of different origin.

Muscle regeneration supports muscle function in aging, and plays a role in the functional impairment caused by progressive neuromuscular diseases. Major substances controlling this process are growth factors and the extracellular matrix (ECM). Thus, follistatin is known to antagonize the function of several members of the TGF-ß family of secreted signaling factors, including myostatin-the most powerful inhibitor of muscle growth characterized to date. Decorin-a small leucine-rich proteoglycan-traps myostatin and modulates its activity towards myogenic cells in the ECM. In addition, there are few reports concerning the regenerative muscle process of masseter muscles, which are of branchial arch origin, in mdx mice. Thus, in order to clarify the muscle regenerative process of masseter muscle, gene and protein expression of myostatin, follistatin and decorin were examined using the tibialis anterior (TA)muscle as a positive control. In both muscles, a gradual increase in mRNA myostatin, follistatin and decorin expression was detected, with the increase being greater in TA muscle than in masseter muscle. At 2 weeks, both muscles exhibited normal skeletal muscle cells. At 3 weeks, masseter muscle demonstrated scant areas of necrosis, whereas large necrotic zones were seen in TA muscle. At 4 weeks, the formation of necrotic tissue and presence of follistatin protein was observed clearly in masseter muscle. This result indicates that follistatin production is stimulated in the presence of necrosis. Interestingly, both muscles showed the same process of muscular formation, but with different time frames, which could be related to muscle origin.

Expression of HGF and IGF-1 during regeneration of masseter muscle in mdx mice.

This study investigated the expression of the growth factors HGF and IGF-1 during the process of muscle regeneration in mdx mice. HGF and IGF-1 are reportedly expressed during the regeneration of muscle tissue in vitro. However, few studies have focused on the role of HGF and IGF-1 during muscle regeneration in mdx mice, which lack expression of the dystrophin gene. In the present study, we examined the expression of HGF and IGF-1 in masseter muscle during muscle regeneration in mdx and B10 (control) mice using histological analysis, immunohistochemistry and Western blotting, as well as examining gene expression by RT-PCR, at 3, 4 and 9 weeks. Mdx mice showed localized HGF and IGF-1 positivity in the cytoplasm of regenerating muscle cells at 3 and 4 weeks, but hardly any reactivity was evident at 9 weeks. The control group was completely negative for IGF-1 at any of the examined time points. Western blotting showed stronger expression of HGF and IGF-1 in mdx mice than in B10 mice at 3 and 4 weeks, but at 9 weeks the expression was absent in both groups. Similar results were obtained using RT-PCR. These present results suggest that HGF and IGF-1 appear to play an important role during regeneration of the masseter muscle in mdx mice.

Expression of myostatin and follistatin in Mdx mice, an animal model for muscular dystrophy.

Follistatin is a functional antagonist of several members of the TGF-beta family of secreted signaling factors, including myostatin, the most powerful inhibitor of muscle growth characterized to date. Myostatin inhibition offers a novel therapeutic strategy for muscular dystrophy by restoring skeletal muscle mass and suppressing the progression of muscle degeneration. To assess the potential benefits of follistatin in treating muscle degenerative diseases, we examined the expression of myostatin and follistatin in Mdx mice, a model for Duchenne muscular dystrophy, and in B10 mice as a control. Our results demonstrated a temporary and coincident expression of follistatin and myostatin in both mouse strains, but this expression was significantly higher in Mdx mice than in B10 mice. The maximum expression of follistatin and myostatin in the presence of restoring necrotic muscle was detected 4 weeks after birth in Mdx mice. Interestingly, during the stage of complete regeneration, the absence of myostatin and follistatin proteins and a marked decrease in the expression of both genes were observed 9 weeks after birth in both mouse strains. These findings suggest that follistatin not only blocks myostatin but also allows other activators to function in muscle development, emphasizing that follistatin could be a very potent molecule in combating muscle loss during dystrophies and muscle ageing, disuse, or denervation.

Increased expression of decorin during the regeneration stage of mdx mouse.

Satellite cells exist in postnatal muscle tissue and constitute the main source of muscle precursor cells for growth and repair. These cells carry out important roles for skeletal muscle formation postnatally during growth of muscle mass as well as damage-induced regenerative processes. Muscle regeneration supports muscle function in aging and has a role in the functional impairment caused by progressive neuromuscular diseases. Major substances controlling this process are growth factors and extracellular matrix. Myostatin, a member of TGF-beta family, was mainly expressed in muscle tissue. Decorin, a member of the small leucine-rich proteoglycan gene family, is composed of a core protein and a dermatan/chondroitin sulfate chain. Recent studies have shown that decorin enhanced the proliferation and differentiation of myogenic cells by suppressing myostatin activity. Thus, decorin appears to be a new molecule in the myostatin signaling pathway and a promising target for treatment of progressive neuromuscular diseases. Therefore, in this study, we examined the localization of decorin as well as myostatin in a muscular dystrophy model in mdx mice and B10 Scott Snells mice as a control to elucidate the differences between decorin and myostatin messages as well as protein distribution. This study revealed increased expression of decorin protein as well as mRNA at the regenerative stage of mdx mice compared to early stages, while only weak expression of decorin was detected in the control mice. Our study contributes to identifying the relationship between decorin and myostatin as well as the development of a therapeutic strategy for progressive neuromuscular diseases.

Myofiber properties of mouse mylohyoid muscle in the growth period.

The mouse mylohyoid muscle belongs to the mastication-related suprahyoid muscle group. It shows a plate-like morphology and forms the mouth floor. There have been no reports on the characteristics of the mouse mylohyoid muscle fibers, and especially on their functional role during ingestion action, and many points remain unclear. We examined the mouse mylohyoid muscle at both the transcriptional and protein levels by RT-PCR, immunohistochemistry, and Western Blotting. MyHC-2b, which is expressed in almost all head and neck muscles and is thought to play a role in rapid mastication movement, was not detected in the mouse mylohyoid muscle. This result suggests that the mouse mylohyoid muscle has a special function and does not directly function during ingestion.

Relationship between function of masticatory muscle in mouse and properties of muscle fibers.

Mammals exhibit marked morphological differences in the muscles surrounding the jaw bone due to differences in eating habits. Furthermore, the myofiber properties of the muscles differ with function. Since the muscles in the oral region have various functions such as eating, swallowing, and speech, it is believed that the functional role of each muscle differs. Therefore, to clarify the functional role of each masticatory muscle, the myofiber properties of the adult mouse masticatory muscles were investigated at the transcriptional level. Expression of MyHC-2b with a fast contraction rate and strong force was frequently noted in the temporal and masseter muscles. This suggests that the temporal and masseter muscles are closely involved in rapid antero-posterior masticatory movement, which is characteristic in mice. Furthermore, expression of MyHC-1 with a low contraction rate and weak continuous force was frequently detected in the lateral pterygoid muscle. This suggests that, in contrast to other masticatory muscles, mouse lateral pterygoid muscle is not involved in fast masticatory movement, but is involved in functions requiring continuous force such as retention of jaw position. This study revealed that muscles with different roles function comprehensively during complicated masticatory movement.

Effects of mechanical stretching on caspase and IGF-1 expression during the proliferation process of myoblasts.

It has been reported that the synthesis, degradation, and metabolism of muscle proteins in myoblasts, as well as the proliferation and differentiation of cells, are influenced by various related to extracellular signaling molecules, such as neural transmitters, growth factors, and hormones, when muscle tissue has been exposed to mechanical stimulation. However, reports regarding the expression of growth factors during mechanical stimulation of myoblasts are few, and many questions remain unanswered. We examined the mRNA expression of insulin-like growth factor 1 (IGF-1) in myoblasts subjected to mechanical stretching in vitro. In addition, apoptosis caused by intracellular stress has been reported to occur during muscle development at the embryonic stage. To clarify the expression of intracellular stress factors, we here investigated related gene expression. Expression of IGF-1 increased in the early stage of cell stretching, followed by a decrease in the late stage. This suggests that mechanical stimulation resulted in an immediate increase in IGF-1 expression, followed by a decrease as cells acclimated to the inducing environment. Caspase was significantly expressed in a stretch group at 12 hours after the beginning of mechanical stimulation, compared with a control group. This suggests that cellular proliferation is also regulated by intracellular stress factors involving the endoplasmic reticulum, mitochondria, and other organelles during the process of muscle proliferation and differentiation.