Voltage-gated sodium channels (Na-v) consist of a pore-forming alpha subunit (Na-v alpha) associated with beta regulatory subunits (Na-v beta). Adult skeletal myocytes primarily express Na(v)1.4 channels. We found, however, using neonatal L6E9 myocytes, that myofibers acquire a Na(v)1.5-cardiac-like phenotype efficiently. Differentiated myotubes elicited faster Na(v)1.5 currents than those recorded from myoblasts. Unlike myoblasts, I-Na recorded in myotubes exhibited an accumulation of inactivation after the application of trains of pulses, due to a slower recovery from inactivation. Since Na-v beta subunits modulate channel gating and pharmacology, the goal of the present work was to study Na-v beta subunits during myogenesis. All four Na-v beta (Na-v beta 1-4) isoforms were present in L6E9 myocytes. While Na-v beta 1-3 subunits were up-regulated by myogenesis, Na-v beta 4 subunits were not. These results show that Na-v beta genes are strongly regulated during muscle differentiation and further support a physiological role for voltage-gated Na+ channels during development and myotube formation. (c) 2008 Elsevier Inc. All rights reserved.