Kimi Watanabe

Differential adaptation of growth and differentiation factor 8/myostatin, fibroblast growth factor 6 and leukemia inhibitory factor in overloaded, regenerating and denervated rat muscles.

Mice genetically deficient in growth and differentiation factor 8 (GDF8/myostatin) had markedly increased muscle fiber numbers and fiber hypertrophy. In the regenerating muscle of mice possessing FGF6 mutation, fiber remodeling was delayed. Although myostatin and FGF6 may be important for the maintenance, regeneration and/or hypertrophy of muscle, little work has been done on the possible role of these proteins in adult muscle in vivo. Using Western blot and immunohistochemical analysis, we investigated, in rats, the distribution of myostatin, FGF6 and LIF proteins between slow- and fast-type muscles, and the adaptive response of these proteins in mechanically overloaded muscles, in regenerating muscles following bupivacaine injection and in denervated muscles after section of the sciatic nerve. The amounts of myostatin and LIF protein were markedly greater in normal slow-type muscles. In the soleus muscle, myostatin and LIF proteins were detected at the site of the myonucleus in both slow-twitch and fast-twitch fibers. In contrast, FGF6 protein was selectively expressed in normal fast-type muscles. Mechanical overloading rapidly enhanced the myostatin and LIF but not FGF6 protein level. In the regenerating muscles, marked diminution of myostatin and FGF6 was observed besides enhancement of LIF. Denervation of fast-type muscles rapidly increased the LIF, but decreased the FGF6 expression. Therefore, the increased expressions of myostatin and LIF play an important role in muscle hypertrophy following mechanical overloading. The marked reduction of FGF6 in the hypertrophied and regenerating muscle would imply that FGF6 regulates muscle differentiation but not proliferation of satellite cells and/or myoblasts.

Calcineurin is a potent regulator for skeletal muscle regeneration by association with NFATc1 and GATA-2.

The molecular signaling pathways involved in regeneration after muscle damage have not been identified. In the present study, we tested the hypothesis that calcineurin, a calcium-regulated phosphatase recently implicated in the signaling of fiber-type conversion and muscle hypertrophy, is required to induce skeletal muscle remodeling. The amount of calcineurin and dephosphorylated nuclear factor of activated T cells c1 (NFATc1) proteins was markedly increased in the regenerating muscle of rats. The amount of calcineurin co-precipitating with NFATc1 and GATA-2, and NFATc1 co-precipitating with GATA-2 gradually increased in the tibialis anterior muscle after bupivacaine injection. Calcineurin protein was present in the proliferating satellite cells labeled with BrdU in the damaged muscle after 4xa0days. In contrast, calcineurin was not detected in the quiescent nonactivating satellite cells expressing Myf-5. At 4xa0days post injection, many macrophages detected in the damaged and regenerating area did not possess calcineurin protein. Calcineurin protein was abundant in many myoblasts and myotubes that expressed MyoD and myogenin at 4 and 6xa0days post injection. In the intact muscle, no immunoreactivity of calcineurin or BrdU was detected in the cell membrane, cytosol or the extracellular connective tissue. In mice, intraperitoneal injection of cyclosporin A, a potent inhibitor of calcineurin, induced extensive inflammation, marked fiber atrophy, the appearance of immature myotubes, and calcification in the regenerating muscle compared with phosphate-buffered saline-administered mice. Thus, calcineurin may have an important role in muscle regeneration in association with NFATc1 and GATA-2.

A possible role for BDNF, NT-4 and TrkB in the spinal cord and muscle of rat subjected to mechanical overload, bupivacaine injection and axotomy.

Neurotrophins play a crucial role in the regulation of survival and the maintenance of specific functions for various populations of neurons. Neurotrophin-4 (NT-4) is most abundant in skeletal muscle, and is thought to promote sciatic nerve sprouting, inhibit agrin-induced acetylcholine receptor (AChR) clustering, evoke postsynaptic potentiation and induce mitochondrial proliferation. Using Western blot analysis, immunoprecipitation and immunohistochemistry, we investigated the distribution of NT-4 in slow- and fast-type muscles. We also tested the adaptive response of this protein in the mechanically overloaded muscle, in the regenerating muscle following bupivacaine injection and in the denervated muscle. Additionally, we investigated whether TrkB phosphorylation in the spinal cord and in the sciatic nerve occurs through the interaction with BDNF or NT-4 when the innervating muscle is damaged. Markedly more NT-4 was expressed in fast-type muscles compared with the slow types. TrkB protein was more frequently observed around the edge of myofibers (neuromuscular junction) of the soleus muscle compared with the gastrocnemius muscle. TrkB tyrosine phosphorylation occurred in the spinal cord but not in the sciatic nerve 24 h after bupivacaine injection of the innervating muscle. At the same time, the amount of TrkB co-precipitating with BDNF was markedly increased in the spinal cord. A rapid activation of TrkB (1-8 h) was also observed in the spinal cord after axotomy,while the amount of TrkB co-precipitating with NT-4 was markedly lower after axotomy. These results indicate that NT-4 is preferentially distributed in fast-type muscles. Furthermore, by interacting with BDNF and NT-4, the TrkB in the spinal cord may be important for the survival of motoneurons and outgrowth of injured peripheral axons following muscle damage.

The adaptive response of MyoD family proteins in overloaded, regenerating and denervated rat muscles

Using Western blot analysis, we investigated whether the amount of myogenic regulatory factors differs in slow-type and fast-type muscles. In addition, we examined the adaptive response of myogenic regulatory factor protein in the overloaded rat muscles by the ablation of synergists, in the regenerating muscles following bupivacaine injection and in the denervated muscle. The amount of myogenin protein in the slow-type muscle was markedly greater. In contrast, the proteins MyoD and Myf-5 were selectively accumulated in the fast-type muscles. A gradual down-regulation of MyoD and Myf-5 proteins was detected in the denervated fast-type muscles, but not in the myogenin protein content. A rapid down-regulation of myogenic regulatory factor protein was observed both of the mechanically overloaded and in the regenerating muscles. These results indicate that the fast-type-specific gene expression in muscle is modulated by MyoD and Myf-5 proteins and suggest that myogenin protein plays an important role in the reconstruction of damaged neuromuscular connections.

The adaptive response of transforming growth factor-β2 and -βRII in the overloaded, regenerating and denervated muscles of rats

Abstract Using a muscle cell line and satellite cell cultures, it has been shown that transforming growth factor-β (TGF-β) has a powerful inhibitory effect on myoblast replication and differentiation. However, little work has been done on the possible role of TGF-β in adult muscle in vivo. Using Western blot and immunohistochemical analyses, we investigated normal distribution of TGF-β2 and TGF-βRII proteins between slow and fast-type muscles, and the adaptive response of these proteins in the mechanically overloaded muscles, in the regenerating muscles following bupivacaine injection and in the denervated muscle after section of sciatic nerve. Slight TGF-β2 immunoreactivity was detected both in slow- and fast-type muscles of mature rat. The amount of TGF-βRII protein was markedly greater in fast-type muscles. In the overloaded muscle, immunohistochemical analysis showed a marked increase in TGF-β2 immunoreactivity in the mononuclear cells (probably endothelial and perithelial or smooth muscle cells of endomysial capillaries) of the extracellular space at 3 and 6 days post surgery. Rapid increase of TGF-β2 protein and concomitant decrease of the receptor (TGF-βRII) were observed in the mechanically overloaded and regenerating muscles. On the other hand, denervation of slow- and fast-type muscles showed a rapid increase in TGF-β2 protein, but did not elicit a concomitant decrease of TGF-βRII. These results indicate that TGF-βRII is preferentially distributed in fast-type muscles. Furthermore, TGF-β2 may play an important role in muscle hypertrophy and regeneration by the usage of TGF-βRII.

Postnatal profiles of myogenic regulatory factors and the receptors of TGF-β2, LIF and IGF-I in the gastrocnemius and rectus femoris muscles of dy mouse

Abstract The dystrophin-deficient mdx mouse presents muscle fiber necrosis but active muscle regeneration, probably due to an extensive recruitment of myogenic regulatory factors (MRF), several growth factors and cytokines, and favorable interaction of satellite cells. In contrast, the laminin α2 (merosin)-deficient dy mouse shows progressive muscle fiber necrosis and ineffective muscle regeneration. Using Western blot and immunohistochemical analyses, we investigated the adaptive changes in MRF, growth factors and cytokines and their receptors in the muscles of dy mice during postnatal growth. The relative volume of MyoD, myogenin and Myf-5 proteins was markedly lower in the gastrocnemius and rectus femoris muscles of dy mice. Transforming growth factor-β2, leukemia inhibitory factor (LIF) and basic fibroblast growth factor were not up-regulated in the muscles of dy mice. The levels of the LIF receptor and insulin-like growth factor-I receptor levels were markedly decreased in the muscles of dy mice during the entire postnatal period observed in this study. Therefore, unlike the situation in mdx mice, the milieu of regeneration following repetitive damage seems to be degraded in the muscles of dy mice.

The reciprocal change of neurotrophin-4 and glial cell line-derived neurotrophic factor protein in the muscles, spinal cord and cerebellum of the dy mouse.

Abstract. Laminin α2 (merosin)-deficient congenital muscular dystrophy (CMD) patients show progressive muscle fiber necrosis and ineffective muscle regeneration, probably due to a lower formation of multinucleated myotubes due to an adhesion defect of myoblasts to each other. Some recent studies found that CMD patients have a white matter disorder and cerebellum atrophy. In the spinal cord of dy mice, a model of CMD, inducible nitric oxide synthase (iNOS) was markedly expressed. Using Western blotting and immunohistochemical analyses, we investigated the levels of neurotrophin-4 (NT-4), brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) in the central nervous system and skeletal muscles of dy mice. In the dy mice, the microtubule-associated protein-2 (MAP-2) protein level was markedly decreased in the Purkinje and granule cells of the cerebellum, and in lumbar motoneurons of the spinal cord. The motoneurons and axons of dy mice possessed lower expressions of phosphorylated tau. The amount of NT-4 was markedly lower in the cerebellum, spinal cord and hindlimb muscles of dy mice. In dy mice, GDNF was markedly enhanced in the Purkinje and granule cells of the cerebellum, in many lumbar motoneurons, and in the regenerating atrophied fibers. The CNTF protein level did not differ in the hindlimb muscles between the normal and dy mice. Therefore, GDNF could act to inhibit the death of Purkinje and granular neurons, and motoneurons, and to promote the remodeling of the neuromuscular junction of atrophied muscle fibers of dy mice. Furthermore, dy mice include neurogenic abnormalities in the cerebellum and spinal cord along with myogenic disorder of muscle fibers.

Muscular Dystrophy: Centronucleation May Reflect a Compensatory Activation of Defective Myonuclei

Muscular dystrophy has long been believed to be characterized by degeneration and abortive regeneration of muscle fibers (the muscle degeneration theory), but unfortunately its pathogenesis is still unclear and an effective treatment has yet to be developed. As a challenge to the theory, we have proposed an alternative muscle-defective-growth theory and a further bone muscle growth imbalance hypothesis supposing possible defects in bone-growth-dependent muscle growth based on our findings in hereditary dystrophic dy mice (C57BL/6J dy/dy). This review presents some new insights into the pathogenesis of the disease along with our hypothesis, focusing on the physiological meaning of centronucleation, one of the major pathological changes commonly observed in dystrophic muscles of man and experimental animals.