Ca²⁺-related fluorescence signals and tension responses in slow-twitch and fast-twitch mouse fibers stimulated by APs (16°C). (A and B) Bundles of fibers from soleus muscles (A) and EDL muscles (B) of adult mice, age 7–14 wk, were isolated by dissection, and one fiber within the bundle was micro-injected with either furaptra or mag-fluo-4. ΔF/F_R and tension responses were recorded in response to external stimuli that elicited either one AP (dashed traces) or five APs at 67 Hz (continuous traces); the responses to both types of stimulation are shown superimposed. All traces represent averaged responses from several experiments in which movement artifacts in the ΔF/F_R traces were small. All fast-twitch experiments were performed on fibers injected with furaptra (seven fibers in the measurements with a single AP, four in the measurements with five APs); the fast-twitch fibers are from a region of the EDL muscle thought to be rich in parvalbumin (see Baylor and Hollingworth, 2011). The slow-twitch experiments with a single AP are from 11 experiments (five with furaptra and six with mag-fluo-4); those with five APs are from three experiments (one with furaptra and two with mag-fluo-4). Before inclusion in the average responses, the mag-fluo-4 traces were scaled so that their amplitude matched that expected with furaptra (Hollingworth et al., 2009, 2012). ΔF/F_R, in furaptra fluorescence units, was converted to Δf_CaD and Δ[Ca²⁺] as described in the text. For both fiber types, a value of 1 on the tension calibration bar corresponds to the peak tension response with the five-AP stimulus. With one AP, the time of peak and FDHM of the slow-twitch tension response are 192 and 1,001 ms, respectively; the corresponding values for the fast-twitch tension response are 55 and 215 ms; with the five-AP stimulus, the values are 229 and 993 ms for the slow-twitch response and 107 and 236 ms for the fast-twitch response. (C and D) The top traces show simulated furaptra Δf_CaD waveforms (noise-free traces) and measured Δf_CaD waveforms (traces with noise). The simulated traces were obtained with the multi-compartment model described in Fig. 3 and the text. In the model, the reaction scheme between Ca²⁺ and furaptra includes two pools of furaptra, protein-free and protein-bound (Baylor and Hollingworth, 2007); the protein-bound pool corresponds to the indicator molecules that appear to be bound to myoplasmic constituents of low mobility (Konishi et al., 1991). The bottom traces show the SR Ca²⁺ fluxes used to drive the simulations. Even though the measurements were made with the fibers stretched to a long sarcomere length (mean value, 3.7 µm), the experimental Δf_CaD traces may still be slightly contaminated with a small movement artifact.