Mibefradil (Ro 40-5967) inhibits several Ca2+ and K+ currents in human fusion-competent myoblasts

Abstract

1. The effect of mibefradil (Ro 40-5967), an inhibitor of T-type Ca2+ current (I(Ca)(T)), on myoblast fusion and on several voltage-gated currents expressed by fusion-competent myoblasts was examined. 2. At a concentration of 5 microM, mibefradil decreases myoblast fusion by 57%. At this concentration, the peak amplitudes of I(Ca)(T) and L-type Ca2+ current (I(Ca)(L)) measured in fusion-competent myoblasts are reduced by 95 and 80%, respectively. The IC50 of mibefradil for I(Ca)(T) and I(Ca)(L) are 0.7 and 2 microM, respectively. 3. At low concentrations, mibefradil increased the amplitude of I(Ca)(L) with respect to control. 4. Mibefradil blocked three voltage-gated K+ currents expressed by human fusion-competent myoblasts: a delayed rectifier K+ current, an ether-à-go-go K+ current, and an inward rectifier K+ current, with a respective IC50 of 0.3, 0.7 and 5.6 microM. 5. It is concluded that mibefradil can interfere with myoblast fusion, a mechanism fundamental to muscle growth and repair, and that the interpretation of the effect of mibefradil in a given system should take into account the action of this drug on ionic currents other than Ca2+ currents.

Figure 1

Mibefradil reduces myoblast fusion in primary culture. (A) Myoblast fusion was induced by exposing myoblasts to differentiation medium and the fusion index was evaluated after 30 and 38 h, in control conditions and in sister cultures exposed to 5 μM mibefradil. Mibefradil decreased myoblast fusion significantly (P<0.0001) at the two time points evaluated. (B) Upper photograph: culture fixed after 38 h in differentiation medium (control condition) and stained with haematoxylin. Lower photograph: sister culture after 38 h in differentiation medium containing 5 μM mibefradil. Scale bar is 25 μm.

Figure 2

Effect of mibefradil on Ca²⁺ currents of fusion-competent myoblasts. (A) Recording from a fusion-competent myoblast (membrane capacitance 42 pF) exposed 3 days to differentiation medium. The cell, superfused with a solution containing 10 mM Ba²⁺ (see Methods), was held at −90 mV and stepped for 80 ms to a series of potentials between −70 and +30 mV. Leak current was estimated by adding 1 mM cadmium at the end of the experiment and was subtracted. Peak Ba²⁺ currents (current through T-type Ca²⁺ channels) were plotted as a function of the voltage during a step in the absence and in the presence of 2 μM mibefradil. Inset: Ba²⁺ current recorded during a step to −20 mV with and without 2 μM mibefradil. (B) Fusion-competent myoblast (membrane capacitance 10 pF) exposed 3 days to differentiation medium. Steady-state Ba²⁺ currents (current through mainly L-type Ca²⁺ channels) measured at the end of a 80 ms step and plotted as a function of the voltage during the step, in the absence and in the presence of 2 μM mibefradil. Leak current was estimated as in (A) and subtracted. Inset: Ba²⁺ current recorded during a step to +10 mV in control, in 2 μM mibefradil, and 3 μM nifedipine in addition to mibefradil. (C) Peak Ba²⁺ currents (current through T-type Ca²⁺ channels) recorded during a step to −20 mV and normalized to their amplitude measured in control solution are plotted as a function of mibefradil concentration. The line is a Hill equation (IC₅₀=0.68 μM, Hill coefficient=1.4, n=5). All recordings were from fusion-competent myoblasts cultured 3 days in differentiation medium. A similar experiment was performed with the current through L-type calcium channels. Normalized steady-state Ba²⁺ currents measured at the end of the 80 ms voltage step to +10 mV were plotted as a function of mibefradil concentration and fitted with a Hill equation (IC₅₀=2 μM, Hill coefficient=1.6, n=4 or more); the open triangles represent Ba²⁺ currents potentiated by low concentrations of mibefradil (n=8). Inset: Effect of low concentrations of mibefradil on current through L-type calcium channels. Steady state Ba²⁺ currents (leak subtracted) were measured at the end of the 80 ms step and plotted as a function of the voltage during the step for different mibefradil concentrations.

Figure 3

Mibefradil inhibits different types of K⁺ currents of fusion-competent myoblasts. All experiments were done with fusion-competent myoblasts exposed to differentiation medium for 3 days. (A) A myoblast (membrane capacitance 18 pF) was held steadily at −80 mV and stepped for 500 ms to a series of voltages between −80 and +40 mV. The outward current obtained at the end of the step was plotted as a function of the voltage during a step in the absence and in the presence of 3 μM mibefradil. Leak current was obtained by linear extrapolation from the current-voltage relationship between −80 and −40 mV and subtracted. This cell had no I_h-eag, so that the outward current represents exclusively I_K(DR). Currents recorded from the same cell (during a step to +40 mV) without and with 3 μM mibefradil are shown. (B) Another fusion-competent myoblast (membrane capacitance 17 pF) was held steadily at +40 mV for 3 min, and stepped for 500 ms to a series of voltages between −80  and +40 mV. Symbols represent the amplitude of I_h-eag recorded in the absence and in the presence of 3 μM mibefradil. Leak current was obtained as in (A) and subtracted. Steady-state currents at +40 mV in the absence and in the presence of 3 μM mibefradil are shown (same cell). The dotted line indicates the leak at +40 mV. (C) A fusion-competent myoblast (membrane capacitance 26 pF) expressing I_K(IR) was held steadily at −60 mV and stepped to a series of voltages between −120 and −40 mV for 500 ms. Leak subtracted I_K(IR) currents measured in the absence and in the presence of 10 μM mibefradil were plotted against the voltage during the step. Leak current was obtained by adding 500 μM Ba²⁺ to the bath solution. Inward K⁺ currents recorded from the same cell at −120 mV in the absence and in the presence of 10 μM mibefradil are shown. (D) I_K(DR), I_h-eag, and I_K(IR) normalized to their respective control values are plotted as a function of mibefradil concentration. The three data sets were fitted with a Hill equation.