Development of excitability during the in vitro differentiation of a newly established myogenic cell line

Developmental changes in the membrane electrical properties during the differentiation of a newly established clonal myogenic cell line MC3T3-A1/M13 (M13) derived from newborn mouse calvaria were studied using the conventional intracellular recording method. M13 cells proliferated in vitro with a population doubling time of about 20 hr when they were cultured in alpha-MEM containing 10% newborn bovine serum at 37 degrees C. After they had achieved confluence and stopped growing, myotube formation by fusion of individual postmitotic mononucleated cells took place within 48 hr, and it advanced until 70-80% of the total number of nuclei were incorporated into such myotubes. Mononucleated M13 cells had a resting membrane potential (Em) of -22.5 +/- 1.7 mV (mean +/- S. D.) and responded passively to current stimuli, indicating that they are non-excitable. On the contrary, multinucleated myotubes had EmS ranging from -25 to -70 mV, depending on the stage of their development. Newly fused myotubes had relatively less negative EmS and showed no response, whereas myotubes later in development showed delayed rectification against depolarizing current pulses, proving the development of a voltage-sensitive outward current system. Further, mature myotubes had an Em of -58.5 +/- 3.2 mV and generated fast action potentials having a maximum rate of rise of 315 +/- 11 V/sec and a duration of 3.0 +/- 0.5 msec (measured at half-height). These action potentials were identified as tetrodotoxin-insensitive Na+ spikes. These results indicate that the membrane excitability of the M13 myogenic cell line develops well after the formation of myotubes, with an increase in Em as maturation proceeds.