Fast imaging in two dimensions resolves extensive sources of Ca2+ sparks in frog skeletal muscle.

Ca2+ sparks were monitored by confocal laser-scanning microscopy of fluo-3 at video rates, in fast twitch muscle fibres, stimulated by exposure to caffeine and/or low [Mg2+]. Scanning was in two spatial dimensions (‘2D’) or 2D plus time, at 4 ms per image frame. Sparks were identified in 2D images of normalized fluorescence by an automatic procedure, which also evaluated the event’s location and morphometric parameters. Most sparks were circular, but some were elongated, especially in caffeine. Separation of the spark from circular symmetry was quantified by its eccentricity (length/width – 1). In an internal solution with 0·4 mM [Mg2+], sparks (989 events in 4 cells) had amplitude 0·73, width 1·94 μm, length 2·12 μm and eccentricity not significantly different from 0. Upon application of 1 mM caffeine, length (of 2578 events in the same cells) increased significantly (by 0·41 μm, or 19 %), width increased by 0·18 μm (9 %) and amplitude decreased slightly. The eccentricity became significantly different from 0, and the sparks’ long axis predominantly oriented parallel to the plane of the Z disks. More than 10 % of the events in caffeine had length greater than 4 μm, a relatively flat top, and a sharp termination at both ends of the major axis. Additionally, there was only a weak correlation between eccentricity and amplitude. These properties suggest that the elongated events are produced by simultaneous opening of multiple channels within a junction, rather than anisotropic diffusion of Ca2+ or random overlap of round sparks. Elongated events often increased in eccentricity early in their evolution. Then, most remained elongated during their rise and decay, while others spread spatially in the plane of the Z disks. In 1–2 % of the events, the centre of mass migrated in space, over time, at ≈0·1 μm ms−1. These spatio-temporal features require the involvement of multiple release channels, at spatially resolvable locations. Because sources often spread over distances greater than 1 or 2 μm, and arrays of junctional elements (couplons) are at most 1·2 μm long, it must be possible for activation of release to propagate between neighbouring couplons, especially under the influence of caffeine and/or low [Mg2+]. The process of Ca2+ release for contractile activation in striated muscle requires that dihydropyridine receptors in the transverse tubules sense the action potential, and transduce it to opening of the sarcoplasmic reticulum Ca2+ channels (review by Leong & MacLennan, 1999). In amphibian skeletal muscle, Ca2+ sparks (Cheng et al. 1993; Tsugorka et al. 1995) constitute a distinctive, and apparently dominant form of Ca2+ release (Klein et al. 1996). Although sparks require Ca2+ as mediator (Klein et al. 1996; Shirokova & Rios, 1997; Gonzalez et al. 2000a), little else is known about their mechanism (e.g. Niggli, 1999; Shirokova et al. 1999; Schneider, 1999). Our recent work (Gonzalez et al. 2000a,b) provided evidence that spark sources involve multiple channels, presumably some or all of the channels in a couplon (junctional array of release channels; Stern et al. 1997; Franzini-Armstrong et al. 1999). If this is the case, then spark sources must be extensive, rather than punctual, and sparks may be asymmetric, not spherically symmetric as they are often assumed to be (e.g. Rios et al. 1999). Until now, two-dimensional spatial properties of sparks have been explored by line scanning successively in two directions (Cheng et al. 1996), a method that does not yield two-dimensional information on individual events. (Such information is present, but not systematically analysed, in images of Tanaka et al. 1997.) We reported a sizable increase in spark width under caffeine, using line scanning parallel to the fibre axis (Gonzalez et al. 2000a). This direction is roughly perpendicular to the long axis of the couplon, and hence especially unsuitable for exploring spatial aspects of the sources. To test the possible involvement of extended arrays of channels in spark generation it is necessary to simultaneously monitor the events in two spatial directions. We now take advantage of the speed of a video-rate laser-scanning confocal system to study for the first time the properties of skeletal muscle Ca2+ sparks in two dimensions of space, and follow the evolution of such images in time. It becomes apparent that caffeine, which increases the sensitivity of release channels to stimulation by Ca2+ (e.g. Herrmann-Frank et al. 1999), causes sparks to grow in the direction of the junctional arrays of release channels.