Triad proteins and intracellular Ca2+ transients during development of human skeletal muscle cells in aneural and innervated cultures.

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.