2.4. Mant-ATP Chase Experiments

On the day of the experiments, bundles were transferred to the relaxing solution and single myofibres were isolated. Their ends were individually clamped to half-split copper meshes designed for electron microscopy (SPI G100 2010C-XA, width, 3 mm), which had been glued to glass slides (Academy, 26 × 76 mm, thickness 1.00–1.20 mm). Cover slips were then attached to the top (using double-sided tape) to create flow chambers (Menzel-Gläser, Braunschweig, Germany, 22 × 22 mm, thickness 0.13–0.16 mm) [18,19]. Subsequently, at 25 °C, for each muscle fibre, the sarcomere length was checked using the brightfield mode of a Zeiss Axio Scope A1 microscope (Jena, Germany). Fibres with a sarcomere length of ~2.50 µm were further subjected to the Mant-ATP chase protocol. Similar to previous studies [18,19], each fibre was first incubated for 5 min with a rigor buffer. A solution containing the rigor buffer with 250 μM Mant-ATP was then flushed and kept in the chamber for 5 min. At the end of this step, another solution made of the rigor buffer with 4 mM ATP was added with simultaneous acquisition of the Mant-ATP chase. For fluorescence acquisition, a Zeiss Axio Scope A1 microscope was used with a Plan-Apochromat 20x/0.8 objective and a Zeiss AxioCam ICm 1 camera. Frames were acquired every 5 s with a 20 ms acquisition/exposure time using a DAPI filter set, images were collected for 5 min. Three regions of each individual myofibre were sampled for fluorescence decay using the ROI manager in ImageJ as previously published [18,19]. The mean background fluorescence intensity was subtracted from the average of the fibre fluorescence intensity (for each image taken). Each time point was then normalized by the fluorescence intensity of the final Mant-ATP image before washout (T = 0). These data were then fit to an unconstrained double exponential decay using Sigmaplot:

where P1 is the amplitude of the initial rapid decay approximating the ‘disordered-relaxed state’ (DRX) with T1 as the time constant for this decay. P2 is the slower second decay approximating the proportion of myosin heads in the ‘super-relaxed state’ (SRX) with its associated time constant T2. To obtain DRX and SRX values, P1 and P2 were adjusted for the level of non-specific binding found to be 40% in skeletal muscle [16].