Indo-1 fluorescence signals elicited by membrane depolarization in enzymatically isolated mouse skeletal muscle fibers

Abstract

Indo-1 fluorescence signals were measured from one extremity of enzymatically isolated skeletal muscle fibers of mice. An original and simple method was developed to allow the measurements to be made under voltage-clamp control: the major part of a single fiber was embedded in silicone grease, so that only a short portion of one end of the fiber, from which the fluorescence measurements were taken, was in contact with the external solution. Membrane potential was held and varied by using a patch-clamp amplifier in whole-cell configuration with a single microelectrode, the tip of which was inserted across the silicone grease within the insulated portion of the fiber. In response to 100-ms depolarizing command pulses to voltages more positive than -40 mV (from a holding potential of -80 mV), clear changes in fluorescence were qualitatively observed to feature a time course of rise and decay expected from a change in intracellular calcium concentration ([Ca2+]i) due to voltage-dependent sarcoplasmic reticulum (SR) calcium release. Although the peak [Ca2+]i elicited by a 100-ms depolarization at 0 or +10 mV varied from fiber to fiber, it could clearly reach a value high enough to saturate Indo-1. The overall results show that this method represents an efficient way of measuring depolarization-induced [Ca2+]i changes in enzymatically dissociated skeletal muscle fibers.