Recordings of action potentials, charge movements, and sarcoplasmic reticulum Ca2+ release in isolated adult zebrafish fast skeletal muscle fibers reveals very fast kinetics of excitation–contraction coupling

Abstract

The zebrafish has emerged as a very relevant animal model to decipher the pathophysiology of human muscle disorders. However, the vast majority of studies on zebrafish skeletal muscle have investigated genetic, histological, and molecular aspects, but functional approaches at the cellular level, especially in the field of excitation-contraction (EC) coupling, are scarcer and generally limited to cultured myotubes or fibers from embryonic zebrafish. Considering that zebrafish undergoes profound metamorphosis during transition from larval to adult stage and that number of muscle pathologies come up at ages far beyond embryonic stages, there is an actual need to investigate EC coupling in fully differentiated zebrafish skeletal muscle. In the present study, we were able to implement current and voltage clamp combined with intracellular Ca2+ measurements using the intracellularly loaded Ca2+ dye indo-1 in enzymatically isolated fast skeletal muscle fibers from 1-yr old zebrafish. Recording of action potentials (AP) in current-clamp conditions revealed very fast kinetics of the repolarization phase of AP. Measurements of intramembrane charge movements in voltage-clamp conditions showed that charge movement density was half that measured in mammalian fibers, but they displayed much faster kinetics. Ca2+ transients elicited by depolarization displayed a voltage-dependent phase of activation and voltage- and time-dependent phase of inactivation. Recording of Ca2+ signals elicited by trains of AP at different rates in current-clamp conditions indicated that Ca2+ signals fused at very high stimulation frequencies with no sign of Ca2+ signal decay for the entire 0.5 s duration of the stimulation, giving evidence that fibers were still able to generate AP and the sarcoplasmic reticulum to release Ca2+ with stimulation rates as high as 200 Hz. These data indicate that adult zebrafish fast skeletal muscle fibers exhibit strikingly fast kinetics of EC coupling from AP firing to charge movements and sarcoplasmic reticulum Ca2+ release.