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doi: 10.1242/10.1242/dev.00562

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Acceleration of human myoblast fusion by depolarization: graded Ca²⁺ signals involved

Jian-Hui Liu^1,2, Stéphane König^1,*, Marlène Michel^1,*, Serge Arnaudeau¹, Jacqueline Fischer-Lougheed¹, Charles R. Bader² and Laurent Bernheim^1,

¹ Département de Physiologie, Centre Médical Universitaire, Hôpital Cantonal Universitaire, CH-1211 Geneva 4, Switzerland
² Division de Recherche Clinique Neuro-Musculaire, Département des Neurosciences, Cliniques et Dermatologie, Hôpital Cantonal Universitaire, CH-1211 Geneva 4, Switzerland

View larger version (24K): [in a new window]   Fig. 5. Membrane potential and Ca2+ influx through window current during myoblast fusion. (A) Model of the events leading to an increase in cytoplasmic Ca2+ concentration in fusion-competent myoblasts. The window current was computed from the mean activation and inactivation parameters of T-type Ca2+ currents measured in 11 fusion-competent myoblasts (Bijlenga et al., 2000). Ca2+ influx increases almost fourfold when the membrane potential is depolarized from -74 to -64 mV. (B) Time course of the cytoplasmic free-Ca2+ concentration in fusion-competent myoblasts evaluated with the `inverse-pericam' fluorescent indicator. Images were recorded every 10 seconds. Control (circles): myoblasts transfected with a bicistronic vector containing only the inverse-pericam. AS-HERG (squares): myoblasts transfected with a bicistronic vector expressing HERG antisense and inverse-pericam. (C) Mean fluorescence ratio increase obtained in control and AS-HERG treated fusion-competent myoblasts.

View larger version (32K): [in a new window]   Fig. 1. HERG transcripts and currents in human myoblasts. (A) Total RNA was subjected to northern blot analysis with an HERG probe. A product of expected size is present in human myoblasts maintained in proliferation medium (0 h) and after different times in differentiation medium. Myoblast fusion begins after 24 hours in differentiation medium and after 36-48 hours maximal fusion is reached (70% of the nuclei are in myotubes). Equal loading of lanes was confirmed by Methylene Blue staining. (B) Dofetilide-sensitive (5 µM) endogenous K+ current (subtraction of the current remaining in the presence of 5 µM dofetilide from the total K+ current) recorded in a fusion-competent myoblast. Whole-cell current traces were elicited in the presence or absence of dofetilide (5 µM) during 1 second steps to various potentials (ranging from -85 mV to +15 mV) from a steady holding potential of -5 mV. The current-to-voltage relationships of the current suppressed by dofetilide are represented. Currents suppressed by dofetilide were measured at the peak. The reversal potential of the recorded current was shifted by -5 mV to adjust it to EK (-35 mV; [K+]out=30 mM). Cell capacitance was 24 pF. (C) Whole-cell K+ currents recorded in a proliferating myoblast transfected with an HERG expression vector in the absence (triangles in the current-to-voltage relationships and `control' traces in the inset) and in the presence of 5 µM dofetilide (squares in the current-to-voltage relationships and `dofetilide' traces in the inset). Same voltage protocol as in B. Cells were co-transfected with pEGFP-N3 (Clontech) to facilitate identification of transfected cells. Cell capacitance was 18 pF.

View larger version (23K): [in a new window]   Fig. 2. HERG channel activity contributes to the resting potential of fusion-competent myoblasts. (A) Resting membrane potential of fusion-competent myoblasts before and after application of 5 µM dofetilide. (B) Examples of dofetilide-sensitive (5 µM) K+ currents recorded in a proliferating myoblast overexpressing HERG. The voltage was held steadily at -4 mV and stepped every 15 s to various potentials ranging from -84 mV to +36 mV for 9 seconds. (C) Peak (triangles) and steady-state (circles) HERG current plotted as a function of the voltage steps. Currents were considered at steady-state after 9 seconds. The reversal potential for K+ ions (EK), the mean resting potential of fusion competent-myoblasts in control conditions (Rp) and the mean resting potential of fusion competent-myoblasts in presence of 5 µM dofetilide (dof) are indicated. The reversal potential of the recorded current was shifted by -4 mV to adjust it to EK (-80 mV; [K+]out=5 mM). Smaller plot shows the peak (triangles) and steady-state (circles) HERG conductance-to-voltage relationships. A reversal potential of -80 mV (EK) was used to calculate conductance.

View larger version (44K): [in a new window]   Fig. 3. Dofetilide and E4031 increase the rate of myoblast fusion. Fusion was induced with differentiation medium. The fusion index is defined as the number of nuclei in myotubes divided by the total number of nuclei counted in a given microscope field. Cultures were fixed for 5 minutes at -20°C with 100% methanol, and stained with Haematoxylin. Nuclei were counted in 20 randomly chosen microscope fields in separate cultures. (A,B) Fusion in control conditions is represented by circles. In sister cultures, dofetilide (A, 10 µM) or E4031 (B, 10 µM) were added to the differentiation medium during the entire duration of experiments (triangles). Error bars are omitted because they are smaller than symbols. Photographs represent Haematoxylin-stained cultures in differentiation medium without (control) and with the drugs. At 24-26 hours, multinucleated myotubes (arrows) were observed only in the cultures treated with either dofetilide (A) or E4031 (B). Scale bars: 40 µm. The smaller plot in A represents the percent of fusion increase with respect to control (0 µM dofetilide) after 18 hours in differentiation medium (100% is the maximum fusion increase) in presence of 0.1, 1 and 5 µM dofetilide.

View larger version (43K): [in a new window]   Fig. 4. HERG antisense depolarizes fusion-competent myoblasts and increases the rate of fusion. (A) Control: myoblasts transfected with a bicistronic vector containing only EGFP. AS-HERG: myoblasts transfected with a bicistronic vector expressing HERG antisense and EGFP. Myoblast transfection and fusion index determination are detailed in the Materials and Methods section. Photographs represent Haematoxylin-stained cultures in differentiation medium. Myotubes (arrows) were observed only in cultures of cells transfected with the HERG antisense vector. Scale bars: 40 µm. (B) Resting membrane potential of fusion-competent myoblasts before (-) and after (+) application of 5 µM dofetilide. Myoblasts were transfected as in A.