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Differentiation of Cultured Myoblasts using Electric Stimulation.    Yumi Kawahara1, Chika Umeda1, Reiko Yoshimoto1, Teruyuki Kajiume2, Katsuko Kataoka2  & Louis Yuge1  (1Graduate School of Health Sciences, 2Graduate School of Biomedical Sciences, Hiroshima University, Japan)

   Muscle growth is facilitated by electric stimulation, and this has been clinically used for treatment of muscle atrophy in space flights.   A rat myoblast cell line, L6 (ATCC.CRL.1458) was used.   The cells were seeded in a culture dish (day 0) and cultured in a Dulbecco’s MEM.   The culture dish became confluent on day 6.   We exposed the cells to electric stimulation (group E) and compared them with a normal culture control (group C).   The condition of the square wave stimulation is 50 V, 2 msec, 30 pulses/min and 5 min/day, and we stimulated the cells on day 7, 9, 11 and 13 in group E.   The cells were cultured up to day 21.   We studied morphological characteristics of the cells and expression of myogenin, a myogenic regulatory factor, by RT-PCR and western blot analysis.   The formation of myotubes occurred earlier and more frequently in group E, than in group C.   Myogenin was expressed earlier and continued longer in group E than group C.   Moreover, we succeeded in differentiation of striated muscle cells showing spontaneous contraction in group E.  We then we examined action potential using a cell physiological technique. The differentiation of myoblasts was accelerated by the application of electric stimulation, and even striated muscle cells were obtained.   These results suggest the possibility that electric stimulation, effectively used in clinical therapy of muscle atrophy, facilitates not only the growth of existing muscle fibers but also the differentiation of myoblasts.