Tanaka H, Furuya T, Kameda N, Kobayashi T, Mizusawa H
Department of Neurology, Tokyo Medical and Dental University School of Medicine, Japan.
J Muscle Res Cell Motil. 2000;21(6):507-26. doi: 10.1023/a:1026561120566.
Dihydropyridine receptors (DHPRs), ryanodine receptors (RyRs), and triadin are major components of triads of mature skeletal muscle and play crucial roles in Ca2+ release in excitation-contraction (E-C) coupling. We investigated the expression and localization of these proteins as well as intracellular Ca2+ transients during development of human muscle cells cultured aneurally and innervated with rat spinal cord. mRNAs encoding skeletal muscle isoforms of the DHPR alpha1 subunit (alpha1S-DHPR), the RyR, and triadin were scarce in myoblasts and increased remarkably after myotube formation. Immunocytochemically, alpha1S-DHPR was expressed after myoblast fusion and localized mainly within the cytoplasmic area of aneural myotubes whereas the cardiac isoform (alpha1C-DHPR) was abundant along the plasma membrane. RyRs and triadin were both detected after myotube formation and colocalized in the cytoplasm of aneural myotubes and innervated muscle fibers. Along the plasma membrane of aneural myotubes, colocalization of alpha1C-DHPR with the RyR was more frequently observed than that of alpha1S-DHPR. In innervated muscle fibers, alpha1S-DHPR and RyR were colocalized first along the plasma membrane and later in the cytoplasmic area and formed regular double rows of cross-striation. The alpha1C-DHPR diminished after innervation. In Ca2+ imaging, spontaneous irregular slow Ca2+ oscillations were observed in aneurally cultured myotubes whereas nerve-driven regular fast oscillations were observed in innervated muscle fibers. Both caffeine and depolarization induced Ca2+ transients in aneurally cultured myotubes and innervated muscle fibers. In aneurally cultured myotubes, depolarization-induced Ca2+ transients were highly dependent on extracellular Ca2+, suggesting immaturity of the Ca2+ release system. This dependence remarkably decreased after innervation. Our present results show that these proteins are expressed differently in aneurally cultured myotubes than in adult skeletal muscle, that Ca2+ release in aneurally cultured myotubes is different from in adult skeletal muscle, and that innervation induces formation of a mature skeletal muscle-like excitation-contraction coupling system in cultured human muscle cells.
二氢吡啶受体（DHPRs）、兰尼碱受体（RyRs）和三联蛋白是成熟骨骼肌三联体的主要组成部分，在兴奋收缩（E-C）偶联的Ca2+释放中起关键作用。我们研究了这些蛋白质在无神经培养和大鼠脊髓支配的人肌肉细胞发育过程中的表达、定位以及细胞内Ca2+瞬变情况。编码DHPRα1亚基（α1S-DHPR）、RyR和三联蛋白的骨骼肌亚型的mRNA在成肌细胞中稀少，在肌管形成后显著增加。免疫细胞化学显示，α1S-DHPR在成肌细胞融合后表达，主要定位于无神经肌管的细胞质区域，而心脏亚型（α1C-DHPR）沿质膜丰富。RyRs和三联蛋白在肌管形成后均被检测到，并共定位于无神经肌管和支配肌纤维的细胞质中。在无神经肌管的质膜上，α1C-DHPR与RyR的共定位比α1S-DHPR更常见。在支配肌纤维中，α1S-DHPR和RyR首先沿质膜共定位，随后在细胞质区域共定位，并形成规则的双排横纹。α1C-DHPR在神经支配后减少。在Ca2+成像中，在无神经培养的肌管中观察到自发的不规则缓慢Ca2+振荡，而在支配肌纤维中观察到神经驱动的规则快速振荡。咖啡因和去极化均在无神经培养的肌管和支配肌纤维中诱导Ca2+瞬变。在无神经培养的肌管中，去极化诱导的Ca2+瞬变高度依赖于细胞外Ca2+，表明Ca2+释放系统不成熟。这种依赖性在神经支配后显著降低。我们目前的结果表明，这些蛋白质在无神经培养的肌管中的表达与成年骨骼肌不同，无神经培养的肌管中的Ca2+释放与成年骨骼肌不同，并且神经支配诱导培养的人肌肉细胞中形成成熟的骨骼肌样兴奋收缩偶联系统。
