The use of the indicator fluo-5N to measure sarcoplasmic reticulum calcium in single muscle fibres of the cane toad

Abstract

1. Single fibres from the lumbrical muscles of the cane toad (Bufo marinus) were incubated in fluo-5N AM for 2 h at 35 degrees C in order to load the indicator into the sarcoplasmic reticulum. Fluo-5N is a low-affinity calcium indicator (K(Ca) 90 microM). Successful sarcoplasmic reticulum (SR) loading was indicated by a fluorescence signal that declined during contraction. 2. Confocal microscopy showed that the dye loaded principally in lines perpendicular to the long axis of the fibre that repeated each sarcomere. This is consistent with much of the dye residing in the SR. 3. To establish the site of loading, fibres were exposed to 30 mM caffeine in the presence of 20 microM 2,5-di(tert-butyl)1,4-hydroquinone (TBQ, an SR pump inhibitor) which should release most Ca(2+) from the SR; this procedure reduced the fluorescence to 46 +/- 4 % of the control value. To determine how much indicator was in the myoplasm, fibres were exposed to 100 microg ml(-1) saponin which permeabilizes the surface membrane; saponin treatment reduced the fluorescence to 51 +/- 2 % of the control value. 4. During maximally activated tetani (100 Hz stimulation rate, 22 degrees C) the component of signal from the SR declined by 33 +/- 4 %. During relaxation the SR signal recovered in two phases with time constants of 0.38 +/- 0.14 s and 10.1 +/- 1.7 s. Partially activated tetani (30 Hz stimulation rate) showed a smaller SR signal. Application of the SR Ca(2+) pump inhibitor TBQ slowed the rate of recovery of the SR signal. 5. Muscle fatigue was produced by repeated short tetani until tension was reduced to 50 %. The SR signal during the periods between tetani declined steadily and the SR Ca(2+) signal was eventually reduced to 71 +/- 8 % of the control signal. This signal recovered in two phases when the muscle was rested. An initial phase had a time constant of 1.7 +/- 0.2 s so that by 20 s of recovery the SR Ca(2+) signal was 86 +/- 7 % of control; the second phase was slower and by 5 min the SR Ca(2+) signal was back to control values (98 +/- 5 % control). In addition the magnitude of the SR signal decline associated with each tetanus (Delta[Ca(2+)](SR)) declined monotonically throughout fatigue and returned to control after 5 min recovery. 6. This approach can monitor the SR Ca(2+) concentration in normally functioning muscle fibres with good time resolution. The method confirms other approaches that show that the free Ca(2+) available for release in the SR declines during fatigue. This reduction in [Ca(2+)](SR) will contribute to the failure of Ca(2+) delivery to the myofilaments which is an important cause of muscle fatigue.

Figure 1. Distribution of fluo-5N fluorescence in single intact toad skeletal muscle fibres

The fibre was loaded with fluo-5N AM as described in the Methods. A, confocal image of section of a single fibre. Regions of high, medium and low fluorescence are coloured red, yellow and blue, respectively. The vertical bar represents 10 μm. B, line plot of fluo-5N fluorescence intensity (arbitrary units, a.u.) of a small (7 μm) region along the fibre. C, line plot of fluo-5N fluorescence intensity (a.u.) across the fibre.

Figure 2. Effect of SR Ca²⁺ depletion on fibre fluorescence

Upper panel shows fibre fluorescence (% initial level) and lower panel shows isometric tension of a single intact toad skeletal muscle fibre loaded with fluo-5N AM. The fibre was exposed to 20 μm TBQ for 60 s and then 30 mm caffeine was applied as indicated by the bars. The fluorescence between the dashed lines represents the total SR Ca²⁺ signal.

Figure 3. Effects of saponin and high [Ca²⁺] on fibre fluorescence

Fibre fluorescence signal (% initial level) of a single intact toad skeletal muscle fibre loaded with fluo-5N AM, when the cell membrane was disrupted with 100 μg ml⁻¹ saponin in Ringer solution. Note the large fall in fibre fluorescence after the treatment with saponin. EGTA (1 mm) was then applied in Hepes-buffered salt solution with no added Ca²⁺. When the signal became steady, 20 mm Ca²⁺ was added to this solution; note the large rise in the fluorescence. Finally the EGTA solution was reapplied as indicated by the bar and caused a fall in fibre fluorescence.

Figure 4. Composite figure showing various Ca²⁺ signals during a tetanus

A shows myoplasmic [Ca²⁺] from a fibre loaded with indo-1. B i and C show the SR Ca²⁺ signal and isometric tension during a 0.5 s 100 Hz tetanus (simultaneously recorded from another fibre). The SR Ca²⁺ signal is shown as the percentage of total SR Ca²⁺ signal. B ii shows the SR Ca²⁺ signal on a slower time base (30 s on time bar) from a third fibre.

Figure 5. Effects of stimulation frequency, stimulation duration and TBQ on SR Ca²⁺ signal and tension

A, effect of stimulation frequency. The fibre was stimulated either at 100 Hz (continuous line) or at 30 Hz (dashed line) with a 0.5 s duration. B, effect of tetanus duration. The fibre was stimulated at 100 Hz for 0.5, 1.0, 1, 1.5 and 8 s durations. C, effect of TBQ. The fibre was given a 100 Hz (0.5 s) tetanus in the absence of TBQ (continuous line), in the presence of 1 μm TBQ (dashed line) and in the presence of 10 μm TBQ (dotted and dashed line). The SR Ca²⁺ signal is shown as the percentage of total SR Ca²⁺ signal.

Figure 6. Effects of fatiguing stimulation on SR Ca²⁺ signal

A, the fibre loaded with fluo-5N underwent a fatiguing protocol until tension fell to 50 %. The 1st, 2nd and 3rd sets of records were obtained at the start, mid-point and end of the fatigue protocol. The fibre was then allowed to recover for 3 min (last set of records). The SR Ca²⁺ signal shown in the upper part is shown as the percentage of total SR Ca²⁺ signal. The dashed line shows the initial resting level. B, SR Ca²⁺ signal from another fibre showing the final period of fatiguing stimulation and the first 15 s of recovery. The dashed line shows an exponential curve fitted to the first 15 s of recovery.

Figure 7. Average data of SR Ca²⁺ signals and tension during fatigue and recovery

Each column is the mean ±s.e.m. (n = 8). For each panel the bars show data for the 1st tetanus (expressed as 100 %), the 10th tetanus, 4 min of fatigue, the end of fatigue, 20 s recovery and 5 min recovery. A shows the resting SR Ca²⁺ signal (measured before the tetanus and expressed as a percentage of the total SR Ca²⁺ signal); B shows the change in SR Ca²⁺ signal during a tetanus; C shows developed tension. a, not signficantly different to control; b, not significantly different to the 4 min point; c, not significantly different to the end of fatigue. All other comparisons were significantly different (P < 0.05 on one way ANOVA).