Supplementary Material for: Novel Electrical Stimulation Sets the Cultured Myoblast Contractile Function to ‘On’

<i>Objective: </i>In the present study, the effect of electrical stimulation was examined for the ability to induce morphological, physiological, and molecular biological effects on myoblasts during cell differentiation. <i>Methods:</i> L6 rat myoblasts were electrically stimulated by newly developed methods on culture days 6, 8, 10 and 12. <i>Results: </i>This electrical stimulation accelerated the appearance of myotubes, and subsequently produced spontaneously contracting muscle fibers. Measurement of membrane potential showed that the contracting cell had functional ion channels and gap junctional intercellular communication. In the electrically stimulated cells, an enhanced expression of MyoD family and M-cadherin was also observed. Expression of connexin 43 was increased and maintained at a high level in the electrically stimulated cells. <i>Conclusion:</i> This is the first demonstration of in vitro induction of myoblasts in spontaneously contractile muscle fibers by intermittent stimulation. This novel method for induction of myoblast differentiation represents an important advance in cell therapy.