骨格筋再生過程におけるミオシン重鎖 アイソフォームとmyogenin・MyoD発現について

骨格筋再生過程におけるミオシン重鎖(MHC)アイソフォーム発現とmyogenin，MyoDタンパクの発現様式との関連性を検討するために，塩酸ブピバカインを用いてマウスヒラメ筋損傷モデルを作成し，損傷筋の再生過程を組織形態学的に確認すると同時に，再生各段階におけるMHCアイソフォームと，myogeninおよびMyoDタンパク発現を経時的に検索した．本研究における筋損傷は塩酸ブピバカインをマウス(C57BL/10SnSlc)のヒラメ筋に注入することで作成した．組織学的には，塩酸ブピバカイン投与後3日目で筋線維はほとんど消失し，処置後6日目で中心核を有する再生筋線維がかなり出現し，処置後28日目では対照群のものと同程度まで回復した．生化学的分析では，対照群ヒラメ筋はMHCⅠ(34．3±1．7%)とMHCⅡa(65．7±1．7%)で構成されていた．実験群ヒラメ筋ではMHCⅠは処置後14日目まで減少し，その後増加傾向を示し，処置後90日目では36．3±2．9%となった．また，正常ヒラメ筋では検出されない速筋型MHC(MHC Ⅱd，MHC Ⅱb)が処置後3日目から28日目まで検出された．Western blotを用いた分析では，myogeninタンパク正常ヒラメ筋（遅筋）で検出された一方，前脛骨筋（速筋）においては検出できなかった．実験群ヒラメ筋では，myogeninは対照群と比較して処置後３日目より増加し（3．1±0．5），処置後６日目でピークに達した（5．8±0．8）．それからmyogeninタンパクは徐々に減少していったが，処置後90日目においてもなお対照群ヒラメ筋の1．8倍の発現を維持し続けた．一方，MyoDタンパクは正常前脛骨筋において正常ヒラメ筋の3．3倍の発現が認められた．MyoDは処置後３日目で対照群ヒラメ筋と比較して5．4倍になりピークに達した．その後は徐々に減少し始めた．しかし処置後90日目においても2．2倍の発現があった．これらのことから筋の再生過程においては速筋タイプの筋細胞が出現するmyogeninとMyoDは衛星細胞の分化と筋の再生に密接に関係していることが示唆された．

To investigate the precise mechanism of skeletal muscle cell regeneration, the changing pattern ofmyosin heavy chain(MHC)isoforms during the regenerating process was observed with relation to theactivation of myogenin and MyoD. In addition, histopathological observation of the damaged muscles wasperformed throughout the experiment.In this study, muscle damage was induced by intramuscular injection of bupivacaine hydrochloride in thesoleus muscle of mice (C57BL/10SnSc). In the light microscopic observation, muscle cells had almost disappeared at 3 days after bupivacainetreatment with severe inflammatory cell infiltration. At 6 days after treatment, a considerable number ofregenerating muscle cells containing centrally located nuclei appeared in the damaged soleus muscle. At28 days, these regenerating muscle cells showed almost the same appearance as the control muscle cellscontaining subsarcolemmal nuclei, although a small number of muscle cells with central nuclei were stillrecognized.In the biochemical analysis, control soleus muscles contained only MHC I (34.3±1.7 %)and MHC IIa(65.7±1.7 %). In the damaged muscles, MHC I was decreased toward 14 days after treatment, and thengradually increased. At 90 days, the contents of MHC I was finally recovered to 36.3±2.9 %.0 In addition,MHC IId and MHC IIb appeared in the damaged muscle from 3 to 28 days after treatment. However, theyhad disappeared at 90 days.Using western blot analysis, myogenin protein was recognized in the control soleus muscles (slow typemuscle), while the myogenin could not be found in the first type muscle of the anterior tibial muscle. Themyogenin contents increased to about three fold (3.1±0.5)at 3 days after treatment compared withthose of control muscles and reached the maximum level (5.8±0.8)at 6 days after treatment. Then, myogenin contents gradually decreased,although they still remained high (1.8 times)at the end of experiment (90 days after treatment). Incontrast to the myogenin protein, a high level (3.3 times)of MyoD protein was detected in the anteriortibial muscle compared with that of control soleus muscles. In the damaged soleus muscles, MyoDcontents reached a maximum level (5.4 times)at 3 days after treatment compared with that of controlsoleus muscles, and then gradually decreased toward the end of experiment. However, MyoD protein stillremained 2.2 times compared with that of control soleus muscles. These findings described above indicate that, 1)a property of fast type muscle cells appeared in theregenerating muscle cells during the regenerating process, and 2)myogenin and MyoD are closelyrelated to the differentiation of the satellite cells and regeneration of the skeletal muscle cells.