SAKUMA Kunihiro Toyohashi University of Technology, The Research Center of Physical Fitness Sports and Health, Assistant Professor, 体育保健センター, 助教授 (60291176)
KUBO Toshikazu Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Professor, 医学研究科, 教授 (20178031)
|Budget Amount *help
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2005: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2004: ¥1,800,000 (Direct Cost: ¥1,800,000)
Neuregulin(NRG) belongs to the family of multipotent growth-promoting proteins, and mainly present at neuromuscular junctions(NMJs). Furthermore, NRGs have been shown to be one of the most significant factors to accumulate AChR at NMJs. While several studies have been conducted on the roles of NRGs in muscle fiber development, little is known about their roles in muscle regeneration. We hypothesize that NRGs play important roles in NMJ reconstruction and in the regeneration of muscle fibers. There has been no study that tried to prove this hypothesis. Therefore, we investigated changes in NRG expression and the relationship with muscle satellite cells which were one of the significant factors of muscle regeneration in damaged muscles. Besides, we were also interested in the NRG expression of the motoneuron which connects target organs and central nerve, and we examined the simultaneous changes in NRG expression in the spinal cord.
We used 10 week adult Wistar rats. Muscle regeneration o
f tibialis anterior (TA) muscle was induced in one leg by an intramuscular injection of bupivacaine hydrochloride. The TA muscles of injected legs and spinal cords dissected at 1, 2, 4, 6, 10 and 14 days post surgery.
Western blot shows that NRG proteins in the regenerating muscles decrease on the 1st day and increase after the 4th day up to 2 weeks. NRG mRNA in the muscle increases from 2 to 6 post-surgical days. The cellular localizations of AChR-a and NRG are shown, respectively. The expression of NRG was detected in some of quiescent satellite cells expressing c-Met. Several c-Met negative mononuclear cells existing near the membrane of muscle fibers also possessed NRG immunoreactivity in unoperated TA muscle of rats. At 2 days post-injection, the cellular localizations of PCNA and NRG are shown, respectively. In addition, BrdU-positive satellite cells also possessed a NRG protein in the regenerating muscle at 2 days post surgery.
At 6days after damage, marked NRG and AChR-a immunoreactivities were observed in the regenerating NMJs. Furthermore, NRG expression was also detected at the myogenin- positive satellite cells respectively.
Western blot analysis shows the gradual increase in NRG proteins in the spinal cord after the first day following damage. However NRG mRNA in the spinal cord is not influenced after muscle damage.
In conclusion, we showed that when muscle was damaged, NRG proteins increased in the regenerating muscles. Combined with the change of NRG mRNA, it seems that NRG promote regeneration in the damaged muscle fibers. Secondly, we showed that NRG immunoreactivity was detected through the proliferation and differentiation stages using immunofluorescence. Thirdly, when muscles were damaged, motoneurons seemed to be influenced by peripheral damage. Western blotting demonstrated that NRG proteins increased in the spinal cord with time, following damage. Interestingly, we did not observe significant changes in NRG mRNA. We indicate a possibility that the NRG protein was transported retrogradely to the spinal cord in vivo. Less