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Electrical Stimulation of Myoblasts Develops Spontaneously Contracting Muscle Fibers. 

Y. Kawahara^1,3, K. Yamaoka², C. Umeda¹, R. Yoshimoto¹, T. Kajiume², A. Sasaki¹, S.L. Wu¹, K. Kataoka², and L. Yuge^1,3. ¹Graduate School of Health Sciences, ²Graduate School of Biomedical Sciences, ³Space Bio-Laboratories Y. K., Hiroshima University, Japan. 

   Electric stimulation has been clinically used for treatment of muscle atrophy in space flights.  However, the effect of electrical stimulation, or an ability to induce morphological, physiological, and molecular biological effects on myoblasts during cell differentiation, has remained to be elucidated.  L6 rat myoblast cells (IFO50364) were seeded in 100-mm culture dishes and maintained in a high-glucose Dulbecco modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS).  When L6 cells reached confluence on culture day 6, they were transferred into FBS-free medium and then exposed to electrical stimulation via field electrodes (5min) on days 6, 8, 10, and 12 (group E) using a stimulator to pass rectangular current pulses (2.0 ms, 50 V, 0.5 Hz) between two platinum wires placed in the culture medium.  The electrical stimulation accelerated the appearance of myotubes, and subsequently produced spontaneously contracting muscle fibers.  Measurement of membrane potential showed that the contracting L6 cells had functional ion channels and gap junctions.  In the electrically stimulated cells, expression of MyoD family, myogenin and Myf-6, and myosin proteins was enhanced.  Expression of gap junction protein, connexin 43, was increased and maintained at a high level in the electrically stimulated cells by western blot analysis and immunostaining.  The differentiation of myoblasts was accelerated, 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 development of existing muscle fibers but also the differentiation of myoblasts.