Doublet stimulation increases Ca²⁺ 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 Ca²⁺ 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) Ca²⁺ release, binding of Ca²⁺ to cytosolic buffers, and force enhancement in fast-twitch fibers. Single isolated fibers respond to doublet pulses with two clear Ca²⁺ 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 Ca²⁺ between the first three Ca²⁺ spikes of the transient, maintaining a higher overall free Ca²⁺ 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 Ca²⁺ release rates during doublet stimulation and showed that Ca²⁺-dependent inactivation of ryanodine receptor activity is rapid, occurring ≤1ms after initial release. Furthermore, we modeled Ca²⁺ binding to the main intracellular Ca²⁺ buffers of troponin C (TnC), parvalbumin, and the SR Ca²⁺ pump during Ca²⁺ release and found that the main effect of the second response in the doublet is to more rapidly increase the occupation of the second Ca²⁺-binding site on TnC (TnC₂), resulting in earlier activation of force. We conclude that doublet stimulation maintains high cytosolic Ca²⁺ levels for longer in the early phase of the Ca²⁺ response, resulting in faster saturation of TnC₂ with Ca²⁺, faster initiation of cross-bridge cycling, and more rapid force development.