Doublet stimulation increases Ca binding to troponin C to ensure rapid force development in skeletal muscle.

Fast-twitch skeletal muscle fibers are often exposed to motor neuron double discharges (≥200 Hz), which markedly increase both the rate of contraction and the magnitude of the resulting force responses. However, the mechanism responsible for these effects is poorly understood, likely because of technical limitations in previous studies. In this study, we measured cytosolic Ca during doublet activation using the low-affinity indicator Mag-Fluo-4 at high temporal resolution and modeled the effects of doublet stimulation on sarcoplasmic reticulum (SR) Ca release, binding of Ca to cytosolic buffers, and force enhancement in fast-twitch fibers. Single isolated fibers respond to doublet pulses with two clear Ca spikes, at doublet frequencies up to 1 KHz. A 200-Hz doublet at the start of a tetanic stimulation train (70 Hz) decreases the drop in free Ca between the first three Ca spikes of the transient, maintaining a higher overall free Ca level during first 20-30 ms of the response. Doublet stimulation also increased the rate of force development in isolated fast-twitch muscles. We also modeled SR Ca release rates during doublet stimulation and showed that Ca-dependent inactivation of ryanodine receptor activity is rapid, occurring ≤1ms after initial release. Furthermore, we modeled Ca binding to the main intracellular Ca buffers of troponin C (TnC), parvalbumin, and the SR Ca pump during Ca release and found that the main effect of the second response in the doublet is to more rapidly increase the occupation of the second Ca-binding site on TnC (TnC), resulting in earlier activation of force. We conclude that doublet stimulation maintains high cytosolic Ca levels for longer in the early phase of the Ca response, resulting in faster saturation of TnC with Ca, faster initiation of cross-bridge cycling, and more rapid force development.