Spark- and ember-like elementary Ca2+ release events in skinned fibres of adult mammalian skeletal muscle

Abstract

1. Using laser scanning confocal microscopy, we show for the first time elementary Ca2+ release events (ECRE) from the sarcoplasmic reticulum in chemically and mechanically skinned fibres from adult mammalian muscle and compare them with ECRE from amphibian skinned fibres. 2. Hundreds of spontaneously occurring events could be measured from individual single skinned mammalian fibres. In addition to spark-like events, we found ember-like events, i.e. long-lasting events of steady amplitude. These two different fundamental release types in mammalian muscle could occur in combination at the same location. 3. The two peaks of the frequency of occurrence for ECRE of mammalian skeletal muscle coincided with the expected locations of the transverse tubular system within the sarcomere, suggesting that ECRE mainly originate at triadic junctions. 4. ECRE in adult mammalian muscle could also be identified at the onset of the global Ca2+ release evoked by membrane depolarisation in mechanically skinned fibres. In addition, the frequency of ECRE was significantly increased by application of 0.5 mM caffeine and reduced by application of 2 mM tetracaine. 5. We conclude that the excitation-contraction coupling process in adult mammalian muscle involves the activation of both spark- and ember-like elementary Ca2+ release events.

Figure 1. Elementary Ca²⁺ release events in adult mammalian skeletal muscle

A, consecutive x–y images of absolute fluorescence from a time series of 100 images (interval, 2 s) showing several discrete Ca²⁺ release events in a chemically (a–d) and a mechanically (e–h) skinned fibre from mouse and a chemically skinned fibre from rat (k–n). In all cases, the intracellular free Ca²⁺ and Mg²⁺ concentration was 100 nm and 0.6 mm, respectively. B, representative linescan images of absolute fluorescence from a chemically skinned fibre from mouse (a) and frog (b) muscle showing that the frequency of ECRE in mammalian muscle was about 1-3 per linescan image, compared with a frequency of about 10 events per linescan image in amphibian muscle. C, frequency histograms of the location of the ECRE maxima relative to the sarcomeric structure of skeletal muscle showing that ECRE mainly occurred at triadic junctions in both mammals (a) and frogs (b), because ECRE were most frequent at the expected locations of the transverse tubular system (TTS) of the respective species.

Figure 2. Linescan images of spontaneous and depolarisation-induced elementary Ca²⁺ release events

A, selected images (width, 10 μm) of normalised fluorescence showing the diversity of spontaneous ECRE in chemically or mechanically skinned mammalian fibres. The trace below each image shows the time course at the centre of the event. Short spark-like events (a–c) and long-lasting steady fluorescence events (ember-like events; d) coexisted with others that appeared to result from superposition of spark- and ember-like events (e–g). Morphological event parameter values for all events (amplitude (F/F₀), FWHM, rise time and FDHM, respectively) were: a: 3.57, 2.46 μm, 7.7 ms, 35.7 ms; b: 2.56, 3.4 μm, 16.9 ms, 46.5 ms; c: 2.51, 2.05 μm, 7.5 ms, 12.1 ms; d: 1.49, 1.5 μm, 256.1 ms, 336.2 ms; e: 2.54, 3.0 μm, 17.3 ms, 43.6 ms; f: 2.14, 1.91 μm, 15.9 ms, 79.9 ms; and g: 2.48, 1.91 μm, 5.85 ms, 80.8 ms. B, Ca²⁺ release induced by depolarisation following replacement of the mammalian internal (high-K⁺) solution with high-Na⁺ internal solution in a mechanically skinned fibre. Lower panels: 4 high-resolution images with representative time courses (marked by arrows) showing that individual ECRE (red circles) can be resolved within the global fluorescence signal at the single triad level.

Figure 3. Morphological event parameters of resting skinned fibres in amphibian and mammalian muscle

Normalised frequency histograms showing the distribution of the four morphological parameters, amplitude, FWHM, rise time and FDHM, of all events in chemically skinned amphibian fibres (green), mechanically skinned mammalian fibres (red) and chemically skinned mammalian fibres (blue). The most remarkable differences were in the rise time and FDHM. The ordinates of the histograms for these temporal parameters were scaled so that events with a long rise time or FDHM could be adequately seen. The histograms shown in the insets display the subset of events for parameter values smaller than 50 ms on the full ordinate scale. The amphibian event set contained no ember-like events (defined as FDHM > 50 ms). In mechanically and chemically skinned mammalian fibres, respectively, 6 % and 33 % of all events were ember-like.

Figure 4. Modulation of ECRE frequency in adult mammalian muscle

In A, the effect of 0.5 mm caffeine on the event frequency in a chemically skinned mammalian and a chemically skinned amphibian fibre is demonstrated in two representative x–y images (70 μm × 50 μm). The mean number of events per image in a xyt-series in these two representative fibres increased from 1.24 ± 0.17 (20 images, mammalian) and 2.23 ± 0.19 (50 images, amphibian) under control conditions to 3.42 ± 0.28 (80 images, mammalian) and 4.78 ± 0.53 (50 images, amphibian) in the presence of caffeine. B, the frequency of ECRE observed in a representative chemically skinned mammalian fibre exposed to a free intracellular Mg²⁺ concentration of 0.4 mm (3.58 ± 0.16 events per image, 100 images) was comparable with that observed in a chemically skinned mammalian fibre in 1 mm free Mg²⁺ (3.54 ± 0.14 events per image, 100 images), showing that 1 mm free intracellular Mg²⁺ was not a limiting factor for the observation of ECRE in skinned fibres. C, the frequency of ECRE was reduced from 1.18 ± 0.18 (100 images) to 0.31 ± 0.05 (100 images) upon application of 2 mm tetracaine in a representative chemically skinned mammalian fibre.