Submitted on June 12, 2007
Accepted on November 12, 2007
Neural agrin controls the maturation of the excitation-contraction coupling mechanism in human myotubes developing in vitro
Elena Bandi1, Marko Jevsek2, Tomaz Mars2, Mihaela Jurdana3, Elena Formaggio4, Marina Sciancalepore3, Guido Fumagalli4, Zoran Grubic2, Fabio Ruzzier3, and Paola Lorenzon3*
1 Dept. of Physiology and Pathology, University of Trieste, Italy; Interdepartmental Centre of Molecular Medicine, University of Trieste, Trieste, Italy
2 Institute of Pathophysiology, University of Ljubljana, Ljubljana, Slovenia
3 Dept. of Physiology and Pathology, University of Trieste, Italy
4 Dept. of Medicine and Public Health, University of Verona, Verona, Italy
* To whom correspondence should be addressed. E-mail: pielle{at}dfp.units.it.
The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: i) aneurally cultured; ii) co-cultured with fetal rat spinal cord explants; iii) aneurally cultured in medium conditioned by co-cultures and iv) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca2+-imaging indicated that co-culturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism. The effect of spinal cord explants was mimicked by treatment with medium conditioned by co-cultures or by addition of 1 nM recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors and dihydropyridine-sensitive L-type Ca2+ channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both ryanodine receptors and L-type Ca2+ channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.
