Follistatin is a functional antagonist of several members of the TGF-β 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.

The mdx mouse, a model of muscular dystrophy, lacks dystrophin, a cell membrane protein. It is known that the lack of dystrophin causes muscle fiber necrosis from 2 weeks after birth, and the majority of necrotic muscle fibers are replaced by regenerated muscle fibers by 4 weeks after birth. A recent study indicated the possibility that mitochondria-mediated intracellular stress, a phenomenon similar to apoptosis, may be produced during muscle fiber necrosis, but did not analyze endoplasmic reticulum-mediated intracellular stress.Therefore, we examined the expression of the caspase-12 gene involved in the endoplasmic reticulum stress pathway and the Bax, caspase-9, and caspase-3 genes involved in the mitochondrial stress pathway in the mdx masseter muscle. We found over-expression of caspase-12 in cells at 2-3 weeks after birth when muscle fiber necrosis was not prominent. This suggests that s tress occurs in the endoplasmic reticulum to maintain cell morphology in the absence of dystrophin. In addition, Bax was abundantly expressed in the mdx masseter muscle at 3 weeks after birth, and the expression of caspase-9 and -3 was prominent at 3-4 weeks after birth when necrosis and regeneration were marked. These results indicate that endoplasmic reticulum and mitochondrial stresses are produced during necrosis of 3 the mdx masseter muscle, and suggest that these events are a phenomenon similar to apoptosis.

We examined expression of four important members of myogenic regulatory factors (MRFs) in the myoblasts both at mRNA and protein levels, which were subjected to mechanical stretching in in vitro condition. Our results showed that MyoD expression existed both in the stretch and in the control group at all time periods of the mechanical stimulus. Myf-5 expressed only at early stage of the stretch group. Although mRNA and protein expressions of myogenin and MRF4 were detected both in the stretch and in the control group at 12 h after the stretching, their expressions were only shown in the stretch group at 24 h after the mechanical stimulus. However, at 36 and 48 h, none of the MRFs examined except MyoD appeared in both groups. Our results suggest that the MRFs are up-regulated upon mechanical stimulus and each member plays a different major role for either proliferation or differentiation of the 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

SummaryTo clarify changes in the muscle fiber properties of the temporal muscle related to the start of masticatory movement, we immunohistochemically investigated myosin heavy chain (MyHC) isoform protein expression using pre-weaning and post-weaning mice. In addition, we examined the expression of a gene coding for those MHC proteins. Immediately after weaning, isoforms with fast and potent contractility were frequent. This suggests that the temporal muscle plays an important role in a marked functional change in the oral cavity from lactation to mastication, contributing to oral function in cooperation with other masticatory muscles.