Voltage-dependent Ca2+ fluxes in skeletal myotubes determined using a removal model analysis

Area of Science:

• Muscle physiology
• Cellular electrophysiology
• Calcium signaling

Background:

• Understanding calcium (Ca2+) dynamics is crucial for muscle function.
• Quantifying Ca2+ fluxes and their voltage dependence in myotubes is essential for elucidating excitation-contraction coupling mechanisms.
• Previous methods for measuring Ca2+ fluxes have limitations in accurately capturing rapid cellular events.

Purpose of the Study:

• To quantify the Ca2+ fluxes underlying Ca2+ transients in myotubes.
• To determine the voltage dependence of these Ca2+ fluxes.
• To utilize a novel 'removal model fit' approach for accurate flux measurement.

Main Methods:

• Utilized mouse C2C12 myotubes, voltage-clamped and loaded with fura-2 and EGTA.
• Simultaneously recorded Ca2+ inward currents and intracellular ratiometric fluorescence transients.
• Applied a 'removal model fit' to the decaying phases of Ca2+-dependent fluorescence signals to determine rate constants for Ca2+ removal components.

Main Results:

• Intracellular Ca2+ release was the dominant Ca2+ input flux in most experiments.
• Ca2+ flux exhibited a peak followed by a tonic phase, with voltage-dependent activation curves.
• The voltage dependence of Ca2+ flux components showed similarities to excitation-contraction coupling in mature mammalian muscle fibers.

Conclusions:

• The 'removal model fit' approach successfully quantifies myoplasmic Ca2+ input flux.
• Intracellular Ca2+ release plays a significant role in Ca2+ transients during depolarization.
• The findings support the validity of optical indicator measurements for determining Ca2+ flux dynamics in muscle cells.