Introduction: Fast-twitch skeletal muscle fibres are often exposed to high frequency motorneuron double discharges which are reported to increase both the rate of contraction and the magnitude of the resulting force responses. Previous findings have suggested that an initial 200 Hz doublet action potential at the start of tetanic stimulation enhances force production by significantly increasing the amplitude of initial sarcoplasmic reticulum (SR) Ca2+ release compared to controls (no doublet, Cheng et al., 2013). However, limitations in previous tracking of cytosolic Ca2+ ([Ca2+]c) due to the use of a high-affinity Ca2+ indicator and insufficient temporal resolution (500 Hz) have led to a distorted view of the tetanic Ca2+ response in previous reports (Cheng et al., 2013). Consequently, the relationship between doublet stimulation, the resulting changes in [Ca2+]c and rates of force development are unlikely to have been accurately determined. The aims of this study were to accurately track [Ca2+]c during doublet stimulation in isolated fast–twitch fibres and to use this data to model the effects of doublet-induced SR Ca2+ release on intracellular Ca2+ binding and force enhancement in fast twitch fibres
Methods: Mice were euthanased and the interosseous muscles removed. Single interosseous fibres (isolated via collagenase digestion) were loaded with Mag-Fluo-4 (5μM), and maintained in Kreb’s Ringer, containing the myosin inhibitor BTS (100 μM). Fibres were activated by 10 action potentials at 120 Hz with or without (control) an initial 200 Hz doublet action potential. Ca2+ fluorescence was captured at 9 kHz using a Zeiss 5 Live confocal microscope in line-scan mode. Modelling of the effects of doublet stimulation on binding of Ca2+ to cytosolic buffers and force production was undertaken using a similar approach to that of Baylor & Hollingworth (2003).
Results: In this study, 200 Hz doublet stimulation did not significantly alter the amplitudes of the Ca2+ responses. However, doublet stimulation did increase the minimum fluorescence value between Ca2+ transient spikes (MFBCS) by approximately 200% compared to controls (control initial MFBCS: 61.06 ± 2.82% of 6th Ca2+ response; doublet: 111.27 ± 13.19% of 6th response, P<0.05). Modelling of the changes in Ca2+ binding to the main intracellular Ca2+ buffers of troponin, parvalbumin and the SR Ca2+ pump during tetanic Ca2+ release showed 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 troponin C (TnC2), resulting in earlier activation of force.
Conclusion: Doublet stimulation maintains high Ca2+ levels for longer in the early phase of the Ca2+ response, resulting in earlier saturation of TnC2 with Ca2+, faster initiation of cross-bridge cycling, and a more rapid onset of force development.
Baylor SM, Hollingworth S. (2003). Sarcoplasmic reticulum calcium release compared in slow-twitch and fast-twitch fibres of mouse muscle. J Physiol 551, 125-38.
Cheng AJ, Place N, Bruton JD, Holmberg HC, Westerblad H. (2013). Doublet discharge stimulation increases sarcoplasmic reticulum Ca2+ release and improves performance during fatiguing contractions in mouse muscle fibres. J Physiol 591, 3739-48.