Voltage-dependent Ca²⁺ Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis

The purpose of this study was to quantify the Ca²⁺ fluxes underlying Ca²⁺ transients and their voltage dependence in myotubes by using the “removal model fit” approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 μM) and a high concentration of EGTA (15 mM). Ca²⁺ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca²⁺-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca²⁺ buffer (to represent EGTA), and a sequestration mechanism as Ca²⁺ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca²⁺ input fluxes from the measured Ca²⁺ transients during depolarizing pulses. In most experiments, intracellular Ca²⁺ release dominated the Ca²⁺ input flux. In these experiments, the Ca²⁺ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at −16 and −20 mV, respectively, compared with −2 mV for the activation of Ca²⁺ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 μM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca²⁺ input flux closely resembled the L-type Ca²⁺ current, consistent with the absence of SR Ca²⁺ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca²⁺ input flux based on the optical indicator measurements.