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Aquaporin-4 (AQP4) is a selective water channel, which expresses on the plasma membrane of myofibers and regulates the osmotic pressure, energy metabolism and morphological changes in myofibers by modulating water transport across sarcolemma in skeletal muscles. Although the physiological roles of AQP4 have been gradually clarified in skeletal muscles, the regulatory mechanisms of AQP4 expression have been poorly understood in skeletal muscles. Recently, it was reported that the expression of AQP4 decreased in atrophied skeletal muscles following sciatic nerve transection, but not tail-suspension. Therefore, expecting that the nerve supply to myofibers would be one of the major regulatory factors regulating AQP4 expression in skeletal muscles, we investigated whether the expression patterns of AQP4 were changed in skeletal muscles by denervation and subsequent reinnervation. As a result, while the APQ4 expression levels were significantly decreased by sciatic nerve freezing-induced denervation, subsequently the expression levels of AQP4 were fully restored during reinnervation in skeletal muscles (p

Aquaporin-4 (AQP4) is a selective water channel that is located on the plasma membrane of myofibers in skeletal muscle and is bound to α1-syntrophin. It is considered that AQP4 is involved in the modulation of homeostasis in myofibers through the regulation of water transport and osmotic pressure. However, it remains unclear whether AQP4 expression is altered by skeletal muscle hypertrophy to modulate water homeostasis in myofibers. The present study investigated the effect of muscle hypertrophy on the changes in AQP4 and α1-syntrophin expression patterns in myofibers. Novel findings indicated in the present study were as follows: 1) Expression levels of AQP4 and α1-syntrophin were stably maintained in hypertrophied muscles, and 2) AQP4 was not expressed in the myofibers containing the slow-type myosin heavy chain isoform (MHC) with or without the presence of fast-type MHC. The present study suggests that AQP4 may regulate the efficiency of water transport in hypertrophied myofibers through its interaction with α1-syntrophin. In addition, this study suggests that AQP4 expression may be inhibited by a regulatory mechanism activated under physiological conditions that induces the expression of slow-type MHC in skeletal muscles.

For myogenesis, new myotubes are formed by the fusion of differentiated myoblasts. In the sequence of events for myotube formation, intercellular communication through gap junctions composed of connexin 43 (Cx43) plays critical roles in regulating the alignment and fusion of myoblasts in advances of myotube formation in vitro. On the other hand, the relationship between the expression patterns of Cx43 and the process of myotube formation in satellite cells during muscle regeneration in vivo remains poorly understood. The present study investigated the relationship between Cx43 and satellite cells in muscle regeneration in vivo. The expression of Cx43 was detected in skeletal muscles on day 1 post-muscle injury, but not in control muscles. Interestingly, the expression of Cx43 was not localized on the inside of the basement membrane of myofibers in the regenerating muscles. Moreover, although the clusters of differentiated satellite cells, which represent a more advanced stage of myotube formation, were observed on the inside of the basement membrane of myofibers in regenerating muscles, the expression of Cx43 was not localized in the clusters of these satellite cells. Therefore, in the present study, it was suggested that Cx43 may not directly contribute to muscle regeneration via satellite cells.

Aquaporin-4 (AQP4) is a selective water channel mediating water transport across cell membranes in skeletal muscles. Recently, it was noted that AQP4 is one of the key molecules regulating muscle morphology. Indeed, the AQP4 accumulation level was stably maintained in hypertrophied skeletal muscles. On the other hand, whether the AQP4 accumulation level is stably maintained in atrophied muscles remains poorly understood. The present study investigated the changes in the AQP4 accumulation level in the atrophied muscles at 2 weeks after denervation. As a result, the accumulation level of AQP4 in the atrophied muscle was significantly decreased compared with that in the control muscle (p

The transplantation of myogenic cells is a potentially effective therapy for muscular dystrophy. However, this therapy has achieved little success because the diffusion of transplanted myogenic cells is limited. Hepatocyte growth factor (HGF) is one of the primary triggers to induce myogenic cell migration in vitro. However, to our knowledge, whether exogenous HGF can trigger the migration of myogenic cells (i.e. satellite cells) in intact skeletal muscles in vivo has not been reported. We previously reported a novel in vivo real-time imaging method in rat skeletal muscles. Therefore, the present study examined the relationship between exogenous HGF treatment and cell migration in rat intact soleus muscles using this imaging method. As a result, it was indicated that the cell migration velocity was enhanced in response to increasing exogenous HGF concentration in skeletal muscles. Furthermore, the expression of MyoD was induced in satellite cells in response to HGF treatment. We first demonstrated in vivo real-time imaging of cell migration triggered by exogenous HGF in intact soleus muscles. The experimental method used in the present study will be a useful tool to understand further the regulatory mechanism of HGF-induced satellite cell migration in skeletal muscles in vivo.

MyoD, a myogenic regulatory factor, is rapidly expressed in adult skeletal muscles in response to denervation. However, the function(s) of MyoD expressed in denervated muscle has not been adequately elucidated. In vitro, it directly transactivates cyclin-dependent kinase inhibitor p21 (p21) and retinoblastoma protein (Rb), a downstream target of p21. These factors then act to regulate cell cycle withdrawal and antiapoptotic cell death. Using immunohistochemical approaches, we characterized cell types expressing MyoD, p21, and Rb and the relationship among these factors in the myonucleus of denervated muscles. In addition, we quantitatively examined the time course changes and expression patterns among distinct myofiber types of MyoD, p21, and Rb during denervation. Denervation induced MyoD expression in myonuclei and satellite cell nuclei, whereas p21 and Rb were found only in myonuclei. Furthermore, coexpression of MyoD, p21, and Rb was induced in the myonucleus, and quantitative analysis of these factors determined that there was no difference among the three myofiber types. These observations suggest that MyoD may function in myonuclei in response to denervation to protect against denervation-induced apoptosis via perhaps the activation of p21 and Rb, and function of MyoD expressed in satellite cell nuclei may be negatively regulated. The present study provides a molecular basis to further understand the function of MyoD expressed in the myonuclei and satellite cell nuclei of denervated skeletal muscle.

Aim: MyoD, myogenin, proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase inhibitor p21 (p21) proteins are key molecules in inducing the growth of myogenic cells in vitro. However, it has not been determined which cell types express these factors in hypertrophying skeletal muscles in vivo. Methods: Using immunohistochemical techniques, we examined the spatial and temporal expression patterns of MyoD, myogenin, PCNA and p21 proteins in functionally overloaded rat plantaris muscles induced by ablation of the soleus and gastrocnemius muscles. Results: MyoD and myogenin were detected in myonuclei located inside the dystrophin-positive plasma membrane of myofibres, m-cadherin-positive satellite cell nuclei and nuclei located in the interstitial spaces between myofibres on days 1, 3, 5 and 7 post-surgery. Entry of satellite cells into the cell cycle was indicated by the expression of PCNA on day 3 post-surgery, and withdrawal from the cell cycle was observed by the expression of p21 in satellite cell nuclei on day 5 post-surgery. However, the expression of both PCNA and p21 in satellite cell nuclei disappeared on day 7 post-surgery. Conclusion: These results indicate that proliferated satellite cell-derived myoblasts and undefined myogenic cells located in the interstitial spaces may contribute to an increase in myonuclear number and/or hyperplasia. Furthermore, we provide evidence that all of myonuclei, satellite cells and undefined myogenic cells express both MyoD and myogenin proteins. These results suggest that continual expression of MyoD and myogenin proteins in these cells is an essential molecular event which induces the successful hypertrophy of skeletal muscles.

Adult mammalian skeletal muscles possess a stem cell population, called muscle satellite cells. Satellite cells mainly contribute to restoring damaged or diseased muscles. The migration of satellite cells plays an important role in muscle regeneration, and it has been traditionally thought that satellite cell migration is regulated by lamellipodial/filopodial formation. In addition, it has recently been thought that blebbing/amoeboid formation plays an important role in satellite cell migration. On the other hand, the method/mechanism(s) of the migration of satellite cells located within skeletal muscles in vivo has barely been elucidated. However, because, in recent years, in vivo real-time imaging of satellite cell migration in skeletal muscles has been reported, it is expected to markedly expand our understanding of satellite cell migration in vivo. This review will focus on the regulatory mechanism of satellite cell migration in vitro and new insights into in vivo satellite cell mobility using real-time imaging.

The recruitment of satellite cells, which are located between the basement membrane and the plasma membrane in myofibers, is required for myofiber repair after muscle injury or disease. In particular, satellite cell migration has been focused on as a satellite cell response to muscle injury because satellite cell motility has been revealed in cell culture. On the other hand, in situ, it is poorly understood how satellite cell migration is involved in muscle regeneration after injury because in situ it has been technically very difficult to visualize living satellite cells localized within skeletal muscle. In the present study, using quantum dots conjugated to anti-M-cadherin antibody, we attempted the visualization of satellite cells in both intact and injured skeletal muscle of rat in situ. As a result, the present study is the first to demonstrate in situ real-time imaging of satellite cells localized within the skeletal muscle. Moreover, it was indicated that satellite cell migration toward an injured site was induced in injured muscle while spatiotemporal change in satellite cells did not occur in intact muscle. Thus, it was suggested that the satellite cell migration may play important roles in the regulation of muscle regeneration after injury. Moreover, the new method used in the present study will be a useful tool to develop satellite cell-based therapies for muscle injury or disease.

Aim: Neural cell adhesion molecule (NCAM) and M-cadherin are cell adhesion molecules expressed on the surface of skeletal muscle satellite cell (SC). During myogenic morphogenesis, M-cadherin participates in mediating terminal differentiation and fusion of myoblasts by forming a complex with β-catenin and that NCAM contributes to myotube formation by fusion of myoblasts. Hypertrophy and hyperplasia of functionally overloaded skeletal muscle results from the fusion with SCs into the existing myofibres or new myofibre formation by SC–SC fusion. However, the alterations of NCAM, M-cadherin and β-catenin expressions in SCs in response to functional overload have not been investigated. Methods: Using immunohistochemical approaches, we examined the temporal and spatial expression patterns of these factors expressed in SCs during the functional overload of skeletal muscles. Results: Myofibres with SCs showing NCAM+/M-cadherin−, NCAM+/M-cadherin+ or NCAM−/M-cadherin+ were detected in overloaded muscles. The percentage changes of myofibres with SCs showing NCAM+/M-cadherin−, NCAM+/M-cadherin+ or NCAM−/M-cadherin+ were elevated in day-3 post-overloaded muscles, and then only the percentage changes of myofibres with SCs showing NCAM−/M-cadherin+ were significantly increased in day-7 post-overload muscles (P