A repetitive mode of activation of discrete Ca2+ release events (Ca2+ sparks) in frog skeletal muscle fibres

Abstract

1. Ca2+ release events (Ca2+ 'sparks'), which are believed to arise from the opening of a sarcoplasmic reticulum (SR) Ca2+ release channel or a small cluster of such channels that act as a release unit, have been measured in single, frog (Rana pipiens) skeletal muscle fibres. 2. Under conditions of extremely low rates of occurrence of Ca2+ sparks we observed, within individual identified triads, repetitive Ca2+ release events which occurred at a frequency more than 100-fold greater than the prevailing average event rate. Repetitive sparks were recorded during voltage-clamp test depolarizations after a brief (0.3-2 s) repriming interval in fibres held at 0 mV and in chronically depolarized, 'notched' fibres. 3. These repetitive events are likely to arise from the re-opening of the same SR Ca2+ release channel or release unit operating in a repetitive gating mode ('rep-mode'), rather than from the random activation of multiple, independent channels or release units within a triad. A train of rep-mode events thus represents a series of Ca2+ sparks arising from a single location within the fibre. Rep-mode events are activated among different triads in a random manner after brief repriming. The frequency of repetitive events among all identified events during voltage-clamp depolarization to 0 mV after brief repriming was 3.9 +/- 1.3 %. The occurrence of repetitive events was not related to exposure of the fibre to laser illumination. 4. The events observed within a rep-mode train exhibited a relatively uniform amplitude. Analysis of intervals between identified events in triads exhibiting rep-mode trains indicated similar variations of fluorescence as in neighbouring, quiescent triads, suggesting there was not a significant number of small, unidentified events at the triads exhibiting rep-mode activity. 5. The distribution of rep-mode interspark intervals exhibited a paucity of events at short intervals, consistent with the need for recovery from inactivation before activation of the next event in a repetitive train. The mean interspark interval of repetitive sparks during voltage-clamp depolarizations was 88 +/- 5 ms, and was independent of membrane potential. 6. The individual Ca2+ sparks within a rep-mode train were similar in average amplitude and spatiotemporal extent to singly occurring sparks, suggesting a common mechanism for termination of the channel opening(s) underlying both types of events. The average properties of the sparks did not vary during a train. The relative amplitude of a spark within a rep-mode was not correlated with its rise time. 7. Repetitive Ca2+ release events represent a mode of gating of SR Ca2+ release channels which may be significant during long depolarizations and which may be influenced by the biochemical state of the SR ryanodine receptor Ca2+ release channels.

Figure 1. Repetitive Ca²⁺ sparks within a single triad during a voltage-clamp depolarization

A fibre held at 0 mV was reprimed at -90 mV for 1 s, then stepped to -40 mV for 400 ms (test pulse), then returned to -90 mV before stepping back to the holding potential (see pulse protocol above image). Left, confocal linescan image (x vs. t, with x parallel to the fibre axis) showing Ca²⁺ sparks activated during the test pulse, displayed in pseudocolour as ΔF/F. Of the 38 triads within this image, only 6 triads exhibited spark activity during the depolarization, with 5 triads showing a single event, and the 6th triad showing 6 events. These 6 events probably arose from the repetitive activation of a single SR Ca²⁺ release unit (see text). A small elevation of fluorescence occurring between the 4th and 5th events of the 6-event triad was probably an out-of-focus event originating in another triad, and did not fulfill the criteria for identification as a spark. Right, single-triad time course records of ΔF/F, corresponding to the triads identified by arrowheads in the left panel. Note the similarity in the time course of the sparks within the single-event triads as compared with individual events in the multi-event triad. Fibre 012097c; sarcomere length, 3.8 μm.

Figure 2. Random occurrence of repetitive rep-mode sparks in triads during successive test depolarizations

The fibre was reprimed at -90 mV for 300 ms, then given a 400 ms test depolarization to 0 mV, a step back to -90 mV (see pulse protocol above images) and a final return to 0 mV (not shown). A-D represent 4 applications of this protocol in successive linescan images acquired at the same location of the fibre, separated in time by 30 s. In A, no triads exhibited repetitive rep-mode sparks during the depolarization, but in B, 1 triad near the bottom of the image showed such a train of repetitive sparks. In C, 2 different triads exhibited rep-mode activity while in D, yet another triad showed repetitive spark activity. These results demonstrate that the tendency to exhibit repetitive rep-mode sparks is not a fixed property of any particular triad but that rep-mode is activated randomly among triads. Fibre 110596b; sarcomere length, 3.8 μm.

Figure 3. Spontaneous rep-mode spark activity in a chronically depolarized fibre

The images (A-E) represent 5 successive 1 s duration linescan images acquired from the same region of the fibre at 2 s intervals. A single triad (indicated by the green arrowhead) exhibited trains of spark activity in 3 of the 5 images, while the other triads in the image showed much lower average activity and no indication of repetitive events. The single-triad records of ΔF/F for the indicated triad (green arrowhead and record) and the two adjacent triads (red and blue arrowheads and records) are shown above the images. F shows the spatial profiles at the time of the peak of the 24 sparks identified in the active triad, spatially centred at the green arrowhead, demonstrating that most of the sparks are centred at the same spatial line within the images, and suggesting that each spark in the train arises from the same triad. The T-shaped bars on each plot are 0.5-1.0 units of ΔF/F vertically, and 1 μm horizontally. Each identified spark is indicated by a black arrowhead below the green traces above the images in A-E. The red, green and blue arrowheads to the side of the image and the scale bars in E also apply to A-D. Fibre 012097b; sarcomere length, 3.6 μm

Figure 4. Estimation of the contribution of small events during a rep-mode train

A, linescan image showing a single triad (arrowhead labelled 2) exhibiting a rep-mode train consisting of 6 sparks. B, single-triad time courses of ΔF/F exhibiting spark activity within the active triad, or no identified spark activity in 2 triads (arrowheads labelled 1 and 3 in A) adjacent to the active one. C, variance of fluorescence fluctuations (σ²) of the single-triad records presented in B, showing that the identified sparks resulted in a large increase of σ². In contrast, the regions of record 2 between identified sparks had a value for σ² which was similar to ostensibly inactive triads (1 and 3). Fibre 101196a; sarcomere length, 3.2 μm

Figure 5. Amplitude histograms and sequential selection of local elevations of fluorescence and average rep-mode and non-rep-mode sparks in 1 fibre

In A, histograms were constructed by locating the local maximum of fluorescence of single-triad ΔF/F records, zeroing a 14-point region containing this maximum, and then locating the next maximum and repeating the procedure, until > 80% of the record was zeroed (see text). This operation was performed on the 3 single-triad ΔF/F records in Fig. 4B, labelled 1-3. The upper histogram represents triad 2, while the lower histogram represents triads 1 and 3 in Fig. 4. Note that the 6 visually identified sparks in panel 2 of Fig. 4B were identified by the algorithm (represented as the 6 bars between ΔF/F values of 1.4 and 2.3). In contrast, the remaining local elevations within the record were distributed similarly to the histograms of the two adjacent triads (bottom histogram; panels 1 and 3 in Fig. 4) which showed ostensibly no spark activity. B, sequentially selected values of local maximum of fluorescence of single-triad ΔF/F records (above) for the triad exhibiting rep-mode activity (□) and for the two adjacent triads (+ and ×). Note that the selection sequence was based on the largest remaining average value to 2 adjacent points whereas the values plotted correspond to the maximum amplitude of individual points in the selected segment (see text for further details). Thus, the sequence of plotted amplitude values does not decline monotonically with the selection number. The sequence of values for the adjacent triads was shifted along the abscissa by 7 to correspond with the first amplitude value beyond the 6 identified sparks in the sequence for the rep-mode triad. C and D, surface plots of an average Ca²⁺ spark calculated by shifting and superimposing the 6 individual sparks shown in the rep-mode triad of Fig. 4A (C) or of 28 isolated sparks (D) in the same fibre. #, number.

Figure 6. Amplitude histograms and sequential detection of local fluorescence maxima in triads exhibiting rep-mode behaviour and in the adjacent triads

A, histograms were constructed by recording the local maximum of fluorescence of single-triad ΔF/F records as described for Fig. 5 on 16 triads (13 fibres) exhibiting rep-mode activity. The histogram was normalized to the total number of values from all triads. B, the same procedure was applied to the triads adjacent to the rep-mode triads in A (thick line). The thin line represents local fluorescence maxima exclusive of the 87 identified Ca²⁺ sparks of the distribution in A, normalized to its total. C, sequentially detected values of local fluorescence maxima in single-triad ΔF/F records obtained as described for Fig. 5 for the triads exhibiting rep-mode activity (squares connected by lines) and for the two adjacent quiescent triads (+ and ×). The sequence of values for the adjacent triads was shifted along the abscissa by 87, corresponding to the number of identified sparks in the sequence of rep-mode triads. The upper plot (dashed line) represents the result of a simulation of Ca²⁺ sparks arising from a hypothetical Ca²⁺ channel which exhibits a stochastic pattern of opening and closing. See text for details. D, sequential representation of all non-spark amplitude values for all triads in C are presented on an expanded ordinate scale (same symbols as in C). #, number.

Figure 7. Evaluation of the influence of laser illumination on the frequency of observations of rep-mode

A, the relative frequency of occurrence of rep-mode is plotted as a function of the duration of exposure to laser illumination. Each run consisted of the acquisition of 4 successive images at the same scan-line location in the fibre, with each image representing 1 s of linescan recording. B, the relative frequency of occurrence of rep-mode during the course of the experiments. The run number within each experiment was normalized to the final image number. The data in A and B were taken from 17 fibres, including both voltage-clamp repriming and ‘notched’ fibre experiments.

Figure 8. Voltage dependence of interspark intervals

Intervals between individual events within rep-mode trains were determined during voltage-clamp depolarizations to different potentials after brief repriming (○, from 11 fibres), and in chronically depolarized, ‘notched’ fibres (▵, from 6 fibres). In the voltage-clamp experiments, the duration of the test depolarization ranged from 400-600 ms.

Figure 9. Histograms of interspark intervals for voltage- (A) and ligand-activated (B) Ca²⁺ sparks within rep-mode trains

The data are grouped from 11 fibres (A) and 6 fibres (B). The lines represent fits of a single exponential function plus constant to the data, starting at 75 ms. The value of the time constant from the fit was 47 ms in A, 53 ms in B.

Figure 10. Amplitude and rise time of Ca²⁺ sparks as a function of the duration of the preceding interspark interval

A and B show the amplitude of the 2nd spark in an interval between 2 sparks within a rep-mode train for voltage- (A) and ligand-activated (B) sparks. The amplitude is given as ΔF/F normalized to the average amplitude of all sparks in that rep-mode train. C and D, rise time of the 2nd spark in an interval between 2 sparks, for voltage- (C) and ligand-activated (D) sparks. Same fibres as in Fig. 9.

Figure 11. Mean amplitude and rise time of voltage- and ligand-activated Ca²⁺ sparks as a function of time of occurrence during a rep-mode train

A and B, normalized amplitude of the nth spark within a train during a test depolarization plotted against the mean time of occurrence of the nth spark (n = 1, 2, 3 or 4) for voltage- (A) and ligand-activated (B) sparks. The first event within the train is taken to start at 0 ms. C and D, the rise time of the nth spark within a rep-mode train for voltage- (C) and ligand-activated (D) sparks. The points are the mean ±s.e.m. of 34 (A) or 31 (B) sparks from 17 fibres.

Figure 12. Relation between normalized amplitude and rise time of rep-mode Ca²⁺ sparks

The amplitude of each event in a train was normalized to the mean event amplitude in the train. A, distribution of event amplitudes as a function of rise time for voltage-activated events. This graph includes all the rep-mode events obtained in 11 fibres at all the depolarizations using the repriming protocol (n = 195). B, distribution of event amplitudes as a function of rise time for the ligand-activated events (n = 122, 6 fibres). The lines result from a fit of a linear equation (ΔF/F =ax + b, see text for details). There was no correlation between Ca²⁺ spark amplitude and rise time in either of the experimental conditions.