Fig. 2. Hyperpolarization controls the early activation of calcineurin pathway during human myoblast differentiation. Human primary myoblasts were transiently transfected with either 9NFAT-luc or 4RE-luc or 3MEF2-luc plasmids. Luciferase activity is in arbitrary units. Increased level of NFAT transcription factor activity reflects calcineurin activity. (A) Kinetics of activation of NFAT, myogenic bHLH and MEF2 transcription factors. Luciferase extracts were prepared from proliferating myoblasts (GM) and from myoblasts maintained for 1 to 4 days in differentiation medium (DM). (B) Depolarization inhibits calcineurin activation. Calcineurin activity was assessed using NFAT-luc plasmid. Human myoblasts were induced to differentiate for 4 days either in control conditions (DM) or in presence of 10 mM Cs⁺ (Cs), 116 mM high extracellular K⁺ (KCl), or a combination of 7 µM cyclosporin A (CsA) and 5 µM FK506 (FK). (C) Inhibition of calcineurin pathway strongly decreases myogenic bHLH and MEF2 transcription factors activation. Myogenic bHLH and MEF2 activity was assessed using 4RE-luc and 3MEF2-luc plasmid, respectively. Same conditions as in B. Results are expressed as mean±s.e.m.