fast imaging in two dimensions resolves extensive … · gustavo brum, adom gonzález, juliana...

2000;528;419-433 J. Physiol.

Gustavo Brum, Adom González, Juliana Rengifo, Natalia Shirokova and Eduardo Ríos

skeletal muscle sparks in frog2+Fast imaging in two dimensions resolves extensive sources of Ca

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The process of Ca¥ 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 Ca¥

channels (review by Leong & MacLennan, 1999). In

amphibian skeletal muscle, Ca¥ sparks (Cheng et al. 1993;Tsugorka et al. 1995) constitute a distinctive, and apparently

dominant form of Ca¥ release (Klein et al. 1996). Althoughsparks require Ca¥ as mediator (Klein et al. 1996; Shirokova& R� úos, 1997; Gonz�alez et al. 2000a), little else is known

about their mechanism (e.g. Niggli, 1999; Shirokova et al.

1999; Schneider, 1999). Our recent work (Gonz�alez 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. R� úos 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

Journal of Physiology (2000), 528.3, pp.419—433 419

Fast imaging in two dimensions resolves extensive sources of

Ca¥ sparks in frog skeletal muscle

Gustavo Brum, Adom Gonz�alez *, Juliana Rengifo*, Natalia Shirokova*

and Eduardo R� úos *

*Department of Molecular Biophysics and Physiology, Rush University, 1750 W. HarrisonStreet, Chicago, IL 60612, USA and Departamento de Biof� úsica, Facultad de Medicina,

G. Flores 2125, Montevideo, Uruguay

(Received 2 May 2000; accepted after revision 7 July 2000)

1. Ca¥ sparks were monitored by confocal laser-scanning microscopy of fluo-3 at video rates,

in fast twitch muscle fibres, stimulated by exposure to caffeine andÏor low [Mg¥]. 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.

2. Most sparks were circular, but some were elongated, especially in caffeine. Separation of the

spark from circular symmetry was quantified by its eccentricity (lengthÏwidth − 1).

3. In an internal solution with 0·4 mÒ [Mg¥], sparks (989 events in 4 cells) had amplitude 0·73,

width 1·94 ìm, length 2·12 ìm and eccentricity not significantly different from 0. Upon

application of 1 mÒ caffeine, length (of 2578 events in the same cells) increased significantly

(by 0·41 ìm, or 19%), width increased by 0·18 ìm (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.

4. More than 10% of the events in caffeine had length greater than 4 ìm, 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 Ca¥ or random overlap of round sparks.

5. 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 ìm ms¢.

6. 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 ìm, and arrays of junctional elements (couplons) are at most 1·2 ìm long, it must be

possible for activation of release to propagate between neighbouring couplons, especially

under the influence of caffeine andÏor low [Mg¥].

11050

Keywords:



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 (Gonz�alez 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 Ca¥ 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 Ca¥

(e.g. Herrmann-Frank et al. 1999), causes sparks to grow in

the direction of the junctional arrays of release channels.

METHODS

Experiments were carried out in cut skeletal muscle fibres from the

semitendinosus muscle of Rana pipiens. Following a protocol

approved by our Institutional Animal Care and Use Committee,

adult frogs were anaesthetized in 15% ethanol, then killed by

pithing. Fibres were mounted on the stage of an inverted

microscope with a video-rate confocal scanner (RCM8000, Nikon

Inc., Melville, NY, USA; Tsien, 1990; Kawanishi et al. 1994;Lacampagne et al. 1999).

The experiments were carried out in permeabilized cells, using

methods derived from those of Lacampagne et al. (1998), describedin detail by R� úos et al. (1999). Cells were stretched to between 3·2

and 3·6 ìm per sarcomere and immersed in a solution with low

[Mg¥], which elicited sparks at a frequency of •15 per image. The

solution, adjusted to pH 7 and 260 mosmol kg¢, contained (mÒ):

100 caesium glutamate, 1 EGTA, 5 glucose, 5 phosphocreatine, 10

Hepes, 5 Mg-ATP, 0·1 fluo-3 (Molecular Probes, Eugene, OR,

USA), with added calcium for a nominal [Ca¥] of 100 nÒ, 0·25 mÒ

MgClµ (0·41 mÒ nominal [Mg¥]), plus 8% (40000 kDa) dextran.

Temperature was 17—19°C.

Excitation light of an argon-ion laser (Enterprise 621, Coherent,

Santa Clara, CA, USA) was selected by a 505 nm dichroic mirror

and emitted light by a 515 nm long pass filter. Imaging used a ²40,

1·15 NA water immersion objective (CF UV, Nikon). Scanning, in

two dimensions, proceeded first along what we define as the

y direction, presented vertically in the figures, at a rate of one line

of 512 pixels (spaced 0·167 ìm) every 64 ìs; 481 lines stacked from

left to right in the x direction constitute an image. As lines are

scanned at distances of 0·138 ìm, imaging in the x direction

proceeds at •2·2 ìm ms¢, so that the 15 ìm horizontal calibration

bar in every image corresponds also to about 7 ms. All images

illustrated are presented at equal spacing (of 0·167 ìm), by linear

interpolation in the x direction.

Space- and time-resolved images (termed ‘3D’ for short) were

obtained by scanning repeatedly a 5·3 ìm ² 85 ìm band in the

object, at 4 ms intervals.

Normalization to resting fluorescence

Images of fluorescence F(x, y) or F(x, y, t), and normalized

fluorescence, FÏF0, which provides a first estimate of increase in

local [Ca¥] relative to its resting value, are shown.

Single 2D images do not contain resting fluorescence information in

those locations where sparks occur. Therefore we developed a

procedure, illustrated in Fig. 1, that uses signal averages. Figure 1Ais a full individual 2D image. Up to 30 images could be obtained at

the same location without deterioration or movement. In the

example, 19 images were obtained, each containing several sparks.

Figure 1B is their pixel-to-pixel sum divided by 19. This ‘rough’

average was used as divisor to obtain a rough normalized version of

every individual image. On each the automatic locator identified

spark areas (those exceeding the value 1 by a set amount), and

excised them (zeroing the corresponding pixels) from the original

non-normalized records. Figure 1C is the boxed part of individual

image A after zeroing the spark regions (in red). These excised

images were then added, to obtain the sum image D. To obtain

resting fluorescence, the sum was divided by a (pixel-dependent)

‘counter’ E. The counter is an array, with the dimensions of the

image, whose value at any pixel is the number of images not zeroed

there, and therefore may range between 1 and 19. The ratio of the

sum D and the counter E is the resting fluorescence, F0(x, y),illustrated in Fig. 1F. Normalized images were obtained dividing

F(x, y) by F0(x, y), pixel to pixel. Figure 1G is the image in Anormalized to resting fluorescence. Especially in the presence of

caffeine, among many circular, symmetric sparks, there are some

obviously elongated events. Shown at the bottom is the profile of

normalized fluorescence as a function of coordinate ‘s’ along the

axis of the large event. It made sense to determine the orientation

of this axis relative to the plane of the Z disks.

The direction of Z disks

2D scanning proceeded first in the longitudinal or y direction

(parallel to fibre axis) and rastered transversely, in the x direction.

While the x direction was generally parallel to the plane of the

Z disks, these could be tilted as much as 30 deg, as shown in Fig. 1F.An automatic method was devised to find locally the direction of

Z disks. The method, illustrated in Fig. 2, uses the clear striation

visible in the averaged image of resting fluorescence (Fig. 2A). When

the event locator described below finds a spark in an individual

image, say, at the position marked by crossbars, it then analyses the

surrounding sub-array of approximately 30 ìm ² 30 ìm. This sub-

array is rotated, relative to the cartesian axes, by 1 deg increments

between −45 and 45 deg. Figure 2B shows the sub-array after

rotation by −25 deg (i.e. clockwise). At every rotation the sub-array

is then averaged along the x coordinate, yielding a function F(y)represented in Fig. 2B. (To avoid edge effects produced by rotation

of a square array, only a central 15 ìm ² 15 ìm box in the sub-

array is averaged.) The root mean square (r.m.s.) value of the

average is high at this particular rotation, because the sarcomeric

striation becomes nearly perpendicular to the y axis. Figure 2Cshows the dependence of the r.m.s. on the rotation angle. The

position of its maximum (−28 deg) determines the angle of the

Z disks relative to the y axis.

Figure 2D shows as short segments the direction of the Z disks at

every position where a spark was found in any of the 19 images

from the same region, demonstrating good agreement between the

method and the direction appraised by eye.

G. Brum, A. Gonz�alez, J. Rengifo, N. Shirokova and E. R� úos J. Physiol. 528.3420



Morphometry of events

A computer procedure described earlier for line scans (Cheng et al.1999), which locates events automatically, was modified to work on

2D images. Events were detected — on images normalized and

filtered — by a relative criterion, fluorescence exceeding F0 by 2·5

standard deviations of the non-spark regions. Figure 3A is one

individual image in the presence of caffeine, after normalization.

At the bottom is the profile of normalized fluorescence along the

slanted axis s. Figure 3B illustrates automatic measurements

performed by the locator on the events detected. For each event a

contour was traced (the border of the region where the detection

threshold is exceeded; in blue). On this contour the program located

the two pixels farthest apart from each other. These points

determine the event’s major axis (central line in red). The spark’s

length is the length of this segment, while its width is the distance

between the two tangents to the contour parallel to the major axis

(in red). These two parameters were used to define an absolute

eccentricity, evaluating separation of the event from circular

Ca¥ sparks in three dimensionsJ. Physiol. 528.3 421

Figure 1. Normalization of sparks in two dimensions

A, fluorescence image (after 9-point median filtering), in a cell exposed to 1 mÒ caffeine. The longitudinal

axis of the fibre is represented vertically (y coordinate); scanning proceeds by vertical lines of pixels and

the area is swept from left to right. The grey scale spans 0 to 255 units of fluorescence. B, rough reference

image; average of A plus 18 other images from the same location (portion shown is boxed in A). The colourscale, in E, spans 0 to 63 units of fluorescence. Normalization of image A by reference B was followed by

application of the automatic event detector, which identified spark areas above a threshold level

(mean + 1·3 s.d.). C, the original image in A with spark areas excised (set to 0) and marked in red. D, sumof all 19 images with spark areas excised. The darker areas are those that had sparks in a greater number of

individual images. E, a counter of sparks, which starts at 19 everywhere and is reduced by one in every

pixel where a spark is excised. The colour table spans the range 0—19. F, the resting fluorescence image

F0(x, y), the result of dividing D by E. Note the clear sarcomeric structure. G, image A divided by resting

fluorescence F0(x, y). At bottom is the profile of normalized fluorescence (averaged for a 0·8 ìm band of 5

pixels) along abscissa s. Identifier: 1203n2, image 69 and sum of images 65 to 83.



symmetry, as (lengthÏwidth − 1). A relative eccentricity (with sign)

is defined later.

The orientation of the spark was quantified, relative to the plane of

the Z disk, by an orientation angle. For this purpose, after locating

a spark, the program reverted to the resting fluorescence image to

determine the local direction of the Z disks as described above. For

the environment of the boxed spark in Fig. 3B, this direction is

represented by the black arrow in the enlargement. The orientation

angle á is measured (counterclockwise) between the Z disk direction

and the long axis of the spark (red arrow in the enlargement).

Statistics

Following every parameter average, in parentheses, is the standard

error of the mean. Significance of differences between averages, X1

and Xµ, of samples of sizes N1 and Nµ, was established by ANOVA

or by two-tailed tests of likelihood of the conventionally defined tvariable:

(X1 − Xµ)�{[N1Nµ (N1 + Nµ − 2)]Ï[(N1 + Nµ)(N1S1Â + NµSµÂ)]},

where SjÂ are the sample variances.

RESULTS

Morphology of sparks in two dimensions

2D images of events obtained in the presence of 1 mÒ

caffeine are illustrated in Figs 1 and 3, while images from

the same fibre in the absence of caffeine (reference) are in

Fig. 4. To the eye, most sparks in reference were symmetric,

and approximately circular. This is confirmed by the

contours drawn for every detected event by the locator

program (right side panels on Fig. 4). By contrast, in

caffeine there were many highly asymmetric, oval or

elongated events. This asymmetry might reflect an actual

feature of the local increase in [Ca¥], or trivially result from

anisotropic dye distribution. The asymmetries introduced

by an inhomogeneous distribution of dye were corrected to a

first approximation normalizing F(x, y) by F0(x, y), the

fluorescence at rest, as described in Methods. Figure 1Fshows the resting fluorescence of the boxed portion of


Figure 2. Determination of direction of the Z disks

A, portion of the resting fluorescence image made as described in Fig. 1. The procedure determines the

direction of Z disks at or near the location of a detected spark (marked by the crossbar). A sub-array of

30 ìm ² 30 ìm around this point is rotated incrementally. B, the sub-array after rotation by −25 deg.

Plotted on the right side is F(y), average over the x dimension of the rotated image (to avoid edge effects

clearly visible at the corners of the sub-array, only the portion within the box is used). F(y) varies widelyaround its mean, because the structural striations are almost parallel to the x axis at this rotation angle.

C, root mean square value of the function F(y)-mean, plotted as a function of the rotation angle. Note the

sharp maximum at −28 deg, which is taken to be the local angle of the Z disks. D, resting fluorescence

image with vectors marking the Z disk direction, placed by the spark detector at all locations where events

were found in any of the 19 images. The array of vectors follows correctly the orientation of the Z disks.



Fig. 1A, obtained using 19 images from the same location.

Note that the periodic variation in F0, probably due to dye

accumulation (Tsugorka et al. 1995; Klein et al. 1996),outlines the sarcomeric structure and demonstrates that the

preparation was steady during imaging.

Normalized images in caffeine are shown in Figs 1G and 3A(the lower panels in Fig. 3 are example events from other

images). The periodic pattern of fluorescence present in the

raw images F(x, y) is removed in the normalized images, butthe sparks that were oval originally maintain their shape

upon normalization. Elongated sparks are therefore not a

consequence of uneven dye distribution.

Four spark parameters, defined in Methods, were analysed.

For 13 images in reference (four of which are illustrated in

Fig. 4) and 19 images in caffeine (illustrated in Figs 1 and 3)

the histogram of absolute eccentricity (lengthÏwidth − 1) is


Figure 3. 2D properties and analysis of sparks in caffeine

A, normalized fluorescence image, in a cell exposed to 1 mÒ caffeine. The colour table spans the range

0—2·3. At the bottom is the profile of fluorescence along the abscissa s. B, automatic location and

morphometry of events. The locator finds a contour (blue, see enlargement), a major axis, its length, event

width (distance between tangents parallel to major axis) and orientation angle á between the local direction

of the Z disks (black arrow) and that of the event’s axis (red). Identifier: 1203n2, image 78. C, D and E,example images of large sparks and profiles along their major axis. Identifiers: C, 1203n2.96; D, 0313d.104;E, 1203n2.74.



plotted in Fig. 5A. In both reference and caffeine the

frequency peaks at a small non-zero value (because of noise,

sparks are seldom perfectly symmetric). There is an excess

of events of large eccentricity in caffeine; the difference of

averages is small but significant (see figure legend).

More impressive is the effect of caffeine on the orientation of

sparks relative to the plane of the Z disks. The distribution

of orientation angle á, in Fig. 5B, is nearly isotropic in

reference (dashed line) but becomes concentrated near the

value 0 in caffeine.

A working hypothesis is that the increase in frequency of

elongated events is due to the increase in the number of

channels involved in individual sparks. In this hypothesis,

the increase in event length should occur along junctions,

that is, in the direction of the Z planes. Therefore, the

increase in eccentricity should be accompanied by an

alignment — an increase in the frequency of small angles á.

We combined the two predicted effects, increase in

eccentricity and orientation, into a single indicator, (relative)

eccentricity, with a sign defined as positive when the

orientation is aligned with the Z plane (−45 deg < á


Figure 4. 2D properties of sparks in reference

Left, normalized fluorescence in the fibre of Fig. 1, before exposure to caffeine. Portions represented are

from different images, among 13 obtained in the same location. Right, the locator finds sparks that are

generally circular — the ‘major axis’ is randomly oriented. Identifier: 1203n2, images 7—9, 11.




Figure 5. Distributions of eccentricity and orientation angle

A, histograms of absolute eccentricity, defined as lengthÏwidth − 1. In caffeine (continuous trace) the

distribution is displaced to higher values. The average eccentricity went from 0·36 (s.e.m. = 0·019) in

reference, to 0·43 (s.e.m. = 0·011) in caffeine. The difference is significant by ANOVA (P < 0·03).

B, distribution of orientation angle, which is almost random in reference (dashed), but is concentrated

around 0 in caffeine. C, distribution of relative eccentricity, with sign defined as positive when |á| < 45 deg,

and negative otherwise. In caffeine there are many events with high positive values of eccentricity,

resulting in a clear shift in the positive direction. There are 213 events in reference (detected in 13 images

from the same location), and 409 in caffeine (19 images from the same location). Identifier 1203n2.

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Table 1. Effects of caffeine on spark morphology

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Events Amplitude Width (ìm) Length (ìm) Eccentricity Angle (deg)––––– –––––––– –––––––– –––––––– ––––––––– –––––––––Ref. Caff. Ref. Caff. Ä Ref. Caff. Ä Ref. Caff. Ä Ref. Caff. Ä Ref. Caff. Ä

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

0312b 289 1463 0·49 0·44 −0·1 1·79 1·98 0·19 2·30 2·67 0·37 −0·16 −0·02 0·14 53·9 42·5 −11·4

0313d 206 320 0·59 0·64 0·05 1·83 2·19 0·36 2·31 3·05 0·74 0·00 0·19 0·19 43·0 32·8 −10·2

1203n 281 386 0·81 0·88 0·07 2·20 2·25 0·05 2·94 3·15 0·21 −0·09 0·10 0·19 48·2 35·6 −12·6

1203n2 213 409 1·01 0·82 −0·19 1·92 2·05 0·13 2·59 2·92 0·33 0·04 0·22 0·18 40·6 29·9 −10·7

Total 989 2578 – – – – – – – – – – – – – – –

Mean – – 0·73 0·70 −0·03 1·94 2·12 0·18 2·54 2·95 0·41* −0·05 0·12 0·18* 46·43 35·20 −11·2*

s.e.m. – – 0·12 0·10 0·06 0·09 0·06 0·07 0·15 0·10 0·11 0·05 0·05 0·01 2·95 2·70 0·52

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

Under amplitude, width and length are listed averages of parameter values (determined as described in

Methods). Ref. and Caff., respectively, list averages in reference condition and in the presence of 1 mÒ

caffeine. Angle is the absolute value of the orientation angle á (see Methods). Ä is paired difference of

averages (Caff. − Ref.). Means are averages over all experiments (average of paired differences in bold type).

*Significantly different from 0 (see Methods). Among caffeine effects, the increase in length and

eccentricity and the decrease in angle were statistically significant in every individual experiment.

––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––



< 45 deg) and negative otherwise. The distribution of this

parameter is shown in Fig. 5C. In reference, it was roughly

bell shaped and centred at 0 (mean 0·00, s.e.m. 0·03) as onewould expect from roughly circular sparks with no preferred

orientation. In caffeine the distribution, still bell shaped,

moved positive (mean 0·22 ± 0·02).

In four experiments analysed, nearly 1000 events in

reference and 2600 in caffeine were detected. The average

parameter values are listed in Table 1. Caffeine caused a

significant increase in length, of 16%, and eccentricity,

from an average value of −0·05 in reference, to 0·18. It

increased width by 9%, and it reduced the absolute value of

the orientation angle by 25%. The changes in length,

eccentricity and angle were also significant when examined

in every individual experiment.

Pondering other mechanisms for the production of elongated

sparks, we studied the statistical distribution of other

parameters, illustrated in Fig. 6. As shown in Fig. 6A, the

distribution of amplitude was generally decreasing, after a

mode at low values, which is likely to be determined by

detection limitations rather than reflect an actual property

of the distribution (cf. Cheng et al. 1999). In the example,

1 mÒ caffeine determined a shift of the distribution to lower

amplitudes, by about 0·15 unit of resting fluorescence. A

similar effect of caffeine was reported by Gonz�alez et al.(2000a) using conventional line scanning, and may be

related to a slight increase in resting fluorescence induced

by the drug, rather than a reduction in Ca¥ release.

The distribution of length (shown as probability density in

Fig. 6B and as probability in C) was unremarkable, but forthe fact that caffeine induced a very large increase in the

frequency of long events. Many sparks in caffeine (13% in

the set illustrated) had a length of 4 ìm or more. Two per

cent of them had a length greater than 6 ìm (several are

shown in Fig. 3). Isolated examples were found of events of

up to 15 ìm.


Figure 6. Quantitative properties of sparks in two dimensions

A, histogram of spark amplitude, in reference and caffeine. B, histogram, and C, cumulative histogram of

event length. Note in C that the frequency of events longer than 4 or 6 ìm was 13 and 2%, respectively.

D, histogram of event width. Note abrupt decay of density beyond 2·5 ìm, both in reference and caffeine.

E and F, plots of eccentricity vs. amplitude or width. The correlation coefficient of eccentricity and

amplitude is 0·08, while that of eccentricity and width is 0·04. There are 213 events from 13 images in

reference, 1136 events from 59 images in caffeine. Identifier: 1203n2.



The distribution of widths was interesting. In three of four

fibres examined in reference and caffeine, it showed a

noticeable drop in density at 2·5 to 3 ìm, which could be

taken to indicate a physical barrier to diffusion (the

sarcomere length in all fibres was greater than 3 ìm).

Such a restriction could provide a mechanism for the

production of elongated sparks: Ca¥ coming from a point

source first diffuses isotropically, then reaches an obstacle

and starts to diffuse anisotropically, as water flowing in a

channel or ‘gutter’. This and related mechanisms predict for

elongated sparks a high correlation between eccentricity and

amplitude, and, perhaps most characteristically, a spatial

dependence of fluorescence that peaks at or near the centre

and ‘spills’ to the sides according to the laws of diffusion.

The profiles of fluorescence along the spark axis, shown in

Figs 1 and 3, were not consistent with this hypothesis.

Occasionally, long sparks showed a peak at their centre

(Fig. 3A). Most long sparks, however, had a flat or slanted

top (as in Figs 1G and 3C, D and E). Figure 6E plots

eccentricity vs. amplitude for the sparks in caffeine. Against

the prediction, the variables were not correlated (the

correlation coefficient r Â was on average 0·09, s.e.m. = 0·08,

n = 4 experiments in caffeine). Figure 6F shows that in the

example there was no correlation between width and

eccentricity either (r Â = 0·04, s.e.m. = 0·09, n = 4).

Elongated sparks could also result from random overlap of

circular ones, but this should occur infrequently (as shown

in Discussion). In conclusion, the elongated events

correspond either to activation of an extended source — a

group of channels in an extended area — or to non-random


Figure 7. Sparks in three dimensions

A, normalized fluorescence in reference, imaged at 4 ms intervals in a 5·3 ìm-wide band. B, representativesparks characterized by the automatic locator are circular, and their position is steady. C, circular and oval

sparks coexisting in the same image. D, oval sparks remain elongated during their evolution, although the

earliest image is often more symmetric. For elongated sparks, the major axis is stably oriented, within

Z planes. Identifiers: n1129, images 31 and 41.



overlap of sparks from point sources. Both alternatives

require that multiple channels contribute to the elongated

events in caffeine.

Mobile sparks

The conclusion above is consistent with images, illustrated

in Figs 7—9, obtained with the time series facility of the

RCM8000 system. In this mode a narrow region in the

object is sampled repeatedly at 4 ms intervals, yielding

snapshots of the same spark at different times. These band

images were processed in essentially the same way as full

2D images. Figure 7A illustrates a succession of normalized

images from the same band in reference solution, and Fig. 7Bthe analysis by the locator program. The images feature the

typical evolution of round sparks. As shown in Fig. 7B, thesparks at top and bottom remain roughly circular from

beginning to end; their ‘major axis’, which is only slightly

longer than other diameters, assumes variable orientations,

indicative of basically symmetric events. The sparks remain

in the same location (confirmed by the evolution of their

centre of mass, not shown). Figure 7C, from the same fibre

in reference, features an oval spark with typical evolution.


Figure 8. Propagating sparks

A and D, examples of sparks that invade an adjacent region as they grow (black arrows). B and E, theautomatic locator confirms the expansion, and shows other events that do not move (red arrows),

demonstrating a steady preparation. C and F, evolution of the abscissa of the centres of signal mass of two

sparks in each frame (plots colour-coded to arrows). The event in A appears to grow by recruiting a discrete

spark at time 16 ms, while the one in D grows more continuously. Images are normalized to F0. Identifiers:

0317b, image 130, and n1129, 52.



It starts small and circular, then grows rapidly in length

and remains elongated during most of its development and

decay, with the long axis pointing steadily in the direction

of the Z disk.

A different type of evolution of elongated sparks is

illustrated in Fig. 8. The sparks marked with black arrows

in Fig. 8A and D originate at the edge of the band, then

invade it progressively. The movement is demonstrated by

the evolution of the abscissa of the centre of mass, plotted in

black in Fig. 8C and F. Propagation was real, not an artifactof preparation drift or errant sampling, as witnessed by

‘landmark’ events in the same band (red arrows), which

were steady or moved slowly in the opposite direction.

Because the spreading events were only partially imaged,

whether they actually migrated or simply grew in length is

unclear.

Examples in Fig. 9 demonstrate actual migration of sparks.

The abscissa of the centre of mass of three events in Fig. 9Ais plotted in Fig. 9B, colour-coded to the arrows. ‘Black’ and‘green’ moved at •0·1 ìm ms¢, while other sparks in the

image remained steady. The migrating sparks became

elongated transiently as they moved and grew. The spark

marked by a black arrow in Fig. 9C first invaded the band

from the right edge. Later the peak of the spark moved into

the band as it grew. This evolution is represented three-

dimensionally in the graph of Fig. 9E, plotting the averagein the five central y pixels as a function of x (‘space’) and

time.

Propagating sparks were relatively infrequent. In two

experiments with caffeine, 500 frames with bands were

recorded and 120 were kept for further examination. There

were 410 sparks in the images kept. Twenty-one of those

propagated and had a landmark in the same frame.

Assuming that the records not kept had the same frequency

of events but no propagating sparks, the ratio of

propagatingÏsteady sparks would be 1%. This estimate

increases to about 2% if the requirement for a landmark in

the same frame is waived. We did not attempt to quantify

the frequency of mobile sparks in reference, but it appeared

to be lower, which is consistent with the fact that mobile

sparks tended to be elongated, and with the scarcity of

elongated events in reference.

DISCUSSION

The combination of rapid confocal scanning in two spatial

dimensions and time with automatic detection and

morphometry of large numbers of sparks allowed us to

characterize their spatio-temporal properties without the

limitations imposed by traditional line scanning. Several

new properties of sparks are documented.

In ‘reference’ conditions (which included a low [Mg¥]é, to

elicit a workable frequency of events) sparks were generally

circular in the image plane, reflecting presumably a

spherical three-dimensional shape. This resulted in an

essentially random distribution of the orientation angle, and

a low average eccentricity (Figs 4 and 5, and Table 1). The

average diameter in reference, 1·94 ìm, is substantially

greater than the full width at half-magnitude (FWHM)

usually reported in line scan studies. This is because in the

present work diameters were measured on suprathreshold

contours (Fig. 3) rather than at half-magnitude. Moreover,

in line scans FWHM is measured at the time of peak

fluorescence, while in the present work sparks were caught

at different times, most often after the peak, when spatial

spread continues to increase.

In the presence of caffeine sparks grew in spatial extent (in

agreement with previous observations with line scanning),

but the growth occurred largely within the plane of the

Z disks. Accordingly, the orientation angle concentrated

sharply around the value 0, and the eccentricity increased

and became ‘positive’ — reflecting the increased alignment

of the major axis.

The significance of elongated sparks

Three explanations were considered for elongated sparks.

One, first proposed by Gonz�alez et al. (2000a) in their studyof caffeine effects, is that the increase in frequency of

elongated events is due to a recruitment of multiple

channels to participate together in individual sparks. This

hypothesis justifies the effect of caffeine (as this drug

sensitizes channels to activation by Ca¥, thus facilitating

recruitment) and explains the alignment with the Z planes

that accompanies the increase in eccentricity.

The second alternative is a mechanical constraint that retains

Ca¥ in a ‘gutter’, or, more generally, any anisotropy, localized

or distributed, in the diffusion or binding properties (as

invoked by Cheng et al. 1996, to explain asymmetric sparks

in cardiac muscle). A distributed anisotropy, however, is

against the fact that most sparks are quite symmetric, even

in the presence of caffeine, and that symmetric and

elongated sparks occur some times at the same location in

successive images.

In support of the existence of a discrete barrier to diffusion,

we found an asymmetric distribution of widths, with a

sudden reduction in density at about 2·8 ìm. If this barrier

was the main cause of the elongation of sparks, amplitude

and width should be correlated with eccentricity. Indeed,

eccentricity should increase only upon reaching this

obstacle, which predicts a strong, albeit non-linear

correlation with event width. Amplitude should also be

correlated, because only sparks of a certain amplitude may

reach the barriers. Finally, the profile of concentration

(fluorescence) along the gutter should be characteristic of

diffusion away from an open source.

These predictions were contradicted by the results. There

was no correlation between amplitude and eccentricity

(consistent with the modest decay of spark amplitude with

caffeine), or between width and eccentricity. Perhaps most

significantly, the spatial dependence of fluorescence along





Figure 9. For legend see facing page.



the spark axis was generally flat or slanted rather than

symmetrically decaying from a central maximum.

The third explanation considered was random overlap of

circular sparks, resulting in images with the appearance of

elongated sparks. A first calculation of probability of such

random overlap can be made assuming that all sparks are

equal, so that the occurrence of sparks in contiguous locations

will be sufficient to generate the impression of an elongated

spark. Every image covers about 30 sarcomeres and 62 ìm

in the x direction. The overall frequency of events in caffeine,

•20 per image in the example of Figs 1—4, corresponds to •1

per image per 100 ìm of junction. The relative frequency of

events longer than 4 ìm (requiring overlap of at least two

sparks in a 4 ìm region) is less than the ratio: (conditional

probability of one event occurring in the 4 ìm region

surrounding another)Ï(probability of one event). If events

are independent, then this is equal to the (unconditional)

probability of an event occurring within a 4 ìm region, or

0·04 (1 ² 4 ìmÏ100 ìm), while their actual frequency was

0·13. That of a 6 ìm event (requiring overlap of at least

three sparks) was 0·0016, while their actual frequency was

0·02.

The above calculation may be refined by recognizing that spark

amplitudes and widths are distributed. For example, the long

sparks in Figs 1 and 3 could occur by superposition of round sparks,

of amplitudes between 0·8 and 1·3, and widths between 2 and

3 ìm. These conditions are satisfied by about 15% of the entire

population (e.g. Fig. 6). Taking this condition into account reduces

7-fold the probability of superposition giving rise to 4 ìm sparks,

and 50-fold that of 6 ìm sparks. Further reductions in probability

are required if it is recognized that more than two sparks should

overlap within 4 ìm to really appear as a single 4 ìm event.

When a spark was imaged at different times, radically

different evolutions were found for elongated and circular

ones. While the latter remained circular during their rise

and ebb, the elongated ones had various possibilities. Some

remained elongated, and when this happened, the long axis

remained essentially fixed in the transverse direction. Of

those that remained elongated, some kept growing in

length, invading adjacent areas at •0·1 ìm ms¢. Yet other

events actually moved in space, displacing their peak at a

similar rate.

Therefore, the possible mechanisms at play in the generation

of elongated sparks must involve the activation of multiple

sources (channels or their arrays). The transverse orientation

of their axis suggests recruitment of channels along

junctional arrays. Two chief modalities of recruitment

appear to be at play: a continuum mechanism, whereby

multiple channels are incorporated smoothly to the group

that generates the event (see Figs 7A and 8C and E), and a

discontinuous recruitment of individual channels, or their

(small) groups, each contributing a discrete, more symmetric

spark (see Fig. 8A).

The mechanism of propagation is likely to involve Ca¥-

induced Ca¥ release (CICR) (Endo et al. 1970; Fabiato,

1984). Interestingly, an earlier stochastic model of

propagation of release within couplons, in which a mobile

‘head’ of elevated Ca¥ provides the activation signal, yields

velocities of about 0·1 ìm ms¢, similar to those observed

here (Stern et al. 1997).

The present observations are also relevant to the mechanism

of termination of sparks. Some sparks migrate, implying

that the regions that activate first are also the first to close

(an inactivation, Sham et al. 1998). They, however,

constitute a minority. Sparks that are elongated usually

remain elongated during their decay phase (as in Fig. 7Cand D), which suggests that, if the spark involves a group ofchannels, then the whole activated array must inactivate

more or less simultaneously. This agrees with the conclusion

that release in sparks terminates abruptly, as if coming

from a single channel (Lacampagne et al. 1999). Such rapid

closure of channels was not reproduced by the couplon

model of Stern et al. (1997), in which release stopped by a

Ca¥-mediated inactivation. Allosteric interactions like

those reported by Marx et al. (1998) might provide a faster

mechanism of channel closure.

The increase in length induced by caffeine, or the extent of

observed propagation, should contain information regarding

the spatial extent of the sources. On average, caffeine

increases length of events by 0·41 ìm, a value that is well

within the length of most frog muscle couplons (Franzini-

Armstrong et al. 1999), hence requiring no more than

increased propagation within a couplon. In many cases,


Figure 9. Migrating sparks

A, migrating sparks (green and black arrows), at both ends of a band. B, evolution of abscissa (x) of the

centre of mass for the sparks at the ends of the band (black and green) and one other near the centre (red),

which provides a landmark. The velocity reaches •0·1 ìm ms¢. Identifier: n1129, image 50. C, a spark

(black arrow) first grows towards the centre of the band as its intensity increases; later a local peak becomes

visible and moves in the same direction. A second spark (red arrow) is relatively steady. D, evolution of

abscissa of the spark’s centre of mass. Note that its movement accelerates (to 0·1 ìm ms¢) when the peak

separates from the edge of the band, probably because the centre of mass is better determined when the

whole event is taken into account. E, three-dimensional representation of the evolution of the spark in D.The central five pixels of the spark are averaged at every x position and in every band, then plotted vs. xand time. The plot shows spatial growth first, then movement of the local peak of fluorescence. Identifier

0317b, image 143. Images are normalized to F0.



however, sparks propagate over a micrometre or more (Fig. 9),

and events of several micrometres are easy to find (Figs 1

and 3). Because the length of frog couplons is at most

1·3 ìm, these cases require propagation over multiple

couplons.

The idea that propagation is restricted within one couplon

stems from simulations (Stern et al. 1997), and has

theoretical importance, as it explains why release activation,

an intrinsically self-sustaining process, remains always under

voltage control. The present observations, however, do not

require radically changing this concept. Sparks of multi-

couplon length are infrequent, and it would not be difficult,

just by increasing Ca¥ sensitivity in the model, to simulate

events that occasionally jump between couplons. In keeping

with this idea, there is other evidence of transmission of

activation over micrometre distances, especially in cardiac

muscle (e.g. Parker et al. 1996; Blatter et al. 1997). Bycomparison, skeletal muscle appears much more ‘uptight’ as

regards Ca¥ release. Interestingly, however, loss of control of

Ca¥ release by voltage in the presence of caffeine was

reported (Simon et al. 1989), reflecting perhaps uncontrollablecontagion of CICR under special circumstances.

The effect of event frequency

In the present experiments, the so-called reference

condition was actually non-physiological, altered to have a

finite, albeit low, frequency of spontaneous sparks. This was

done by lowering the free [Mg¥] to about 0·4 mÒ

(Lacampagne et al. 1998). The mechanism of this effect is

thought to be a relief from the inhibition exerted by Mg¥ on

the release channels. Such relief renders the channels more

susceptible to opening, which presumably occurs under the

influence of local Ca¥. On the other hand, caffeine is

believed to increase the sensitivity of channels to activation

by Ca¥, hence their proclivity to open ‘spontaneously’, or be

recruited into a spark. Therefore, both caffeine and low

[Mg¥] should increase the length and eccentricity of sparks,

as both should promote activation, and recruitment of

additional channels.

The study that will test whether the effects of caffeine and

low [Mg¥] are similar, a determination of spark

morphology at different [Mg¥], has not been done. In

support of this idea, however, it appeared that spark length,

eccentricity and orientation grew in parallel with the overall

frequency of events per image. This was seen best when the

frequency of events varied substantially in the course of

time. In one experiment that had eccentricity of 0·00 (0·01)

in reference, while a series of 20 images was being recorded

in the presence of caffeine, the frequency changed

monotonically from 36 to 19 events per image. For the

events in the first 10 images, when frequency averaged 32

events per image, eccentricity was 0·19 (0·02), while in the

second half of the sequence (when the frequency was 23 per

image) the eccentricity was 0·08 (0·02). When the set of

events was divided into four groups, each gathered during

five successive images, eccentricity correlated well with

frequency (r Â was 0·85) but amplitude was essentially

constant (r Â = 0·22).

In conclusion, the parallel evolution of eccentricity and

frequency during uncontrolled changes suggests that the

increase in eccentricity and event length associated with the

presence of caffeine is not specific for this agonist, but a

consequence of an increase in the channels’ propensity to

open.

The nature of ‘standard’ sparks

Sparks that are elongated, or grow in length or move during

their evolution, require the involvement of multiple

channels, opening simultaneously or sequentially. While in

some cases propagation of sparks appears to occur by

discrete jumps, and could be explained by recruitment of a

few channels (or their groups), in others (as in Fig. 9E) it iscontinuously graded, suggesting the involvement of tens of

channels. That the sparks studied here were elicited by non-

physiological stimulants of channel activity may suggest

that the involvement of multiple channels requires such

artificial enhancers, and is not a feature of normal or

standard sparks. On the other hand, a previous study

(Gonz�alez et al. 2000a) showed that caffeine modifies sparks

quantitatively, increasing for instance the frequency of

sparks of large width, rather than inducing qualitatively

different events that do not occur in reference. This suggests

that standard sparks (i.e. those induced by normal stimuli in

physiological internal solutions) should also involve multiple

channels. The same conclusion was reached by us, on

entirely different grounds, in two other publications (R� úos etal. 1999; Gonz�alez et al. 2000b).

Because membrane depolarization also operates by

increasing the propensity of release channels to open (an

effect compared to the removal of Mg¥ inhibition; Lamb,

2000), a similar cooperative activation of channel arrays

might be taking place when the membrane is depolarized,

either experimentally or physiologically during an action

potential. A study of the effect of membrane voltage on the

spatio-temporal morphology of sparks is clearly called for,

and is in progress.

Blatter, L. A., H�user, J. & R�úos, E. (1997). Sarcoplasmic reticulumCa¥ release flux underlying Ca¥ sparks in cardiac muscle.Proceedings of the National Academy of Sciences of the USA 94,4176—4181.

Cheng, H., Lederer, W. J. & Cannell, M. B. (1993). Calciumsparks: elementary events underlying excitation-contractioncoupling in heart muscle. Science 262, 740—744.

Cheng, H., Lederer, M. R., Xiao, R. P., Gomez, A. M., Zhou, Y. Y.,Ziman, B., Spurgeon, H., Lakatta, E. G. & Lederer, W. J.(1996). Excitation-contraction coupling in heart: new insights fromCa¥ sparks. Cell Calcium 20, 129—140.

Cheng, H., Song, L. S., Shirokova, N., Gonz �alez, A., Lakatta,E. G., R� úos, E. & Stern, M. D. (1999). Amplitude distribution ofcalcium sparks in confocal images. Theory and studies with anautomatic detection method. Biophysical Journal 76, 606—617.




Endo, M., Tanaka, M. & Ogawa, Y. (1970). Calcium induced releaseof calcium from the sarcoplasmic reticulum of skinned skeletalmuscle fibres. Nature 228, 34—36.

Fabiato, A. (1984). Dependence of the calcium-induced release formthe sarcoplasmic reticulum of skinned skeletal muscle fibres from thefrog semitendinosus on the rate of change of free Ca¥ at the outersurface of the sarcoplasmic reticulum. Journal of Physiology 353,56P.

Franzini-Armstrong, C., Protasi, F. & Ramesh, V. (1999). Shape,size, and distribution of Ca¥ release units and couplons in skeletaland cardiac muscles. Biophysical Journal 77, 1528—1539.

Gonz�alez, A., Kirsch, W. G., Shirokova, N., Pizarro, G., Stern,M. D. & R�úos, E. (2000a). The spark and its ember. Separately gatedlocal components of Ca¥ release in skeletal muscle. Journal ofGeneral Physiology 115, 139—158.

Gonz�alez, A., Shirokova, N., Kirsch, W. G., Pessah, I. N., Cheng,H., Stern, M. D., Pizarro, G., Brum, G. & R�úos, E. (2000b).Involvement of multiple channels in calcium sparks of skeletalmuscle. Proceedings of the National Academy of Sciences of theUSA 97, 4380—4385.

Herrmann-Frank, A., L�uttgau, H.-C. & Stephenson, D. G. (1999).Caffeine and excitation-contraction coupling in skeletal muscle: astimulating story. Journal of Muscle Research and Cell Motility 20,223—237.

Kawanishi, T., Asou, H., Kato, T., Uneyama, C., Toyoda, K.,Ohata, H., Momose, K. & Takahashi, M. (1994). Ratio-imaging ofcalcium waves in cultured hepatocytes using rapid scanningconfocal microscope and indo-1. Bioimages 2, 7—14.

Klein, M. G., Cheng, H., Santana, L. F., Jiang, Y. H., Lederer,W. J. & Schneider, M. F. (1996). Two mechanisms of quantizedcalcium release in skeletal muscle. Nature 379, 455—458.

Lacampagne, A., Klein, M. G. & Schneider, M. F. (1998).Modulation of the frequency of spontaneous sarcoplasmic reticulumCa¥ release events (Ca¥ sparks) by myoplasmic [Mg¥] in frogskeletal muscle. Journal of General Physiology 111, 207—224.

Lacampagne, A., Ward, C. W., Klein, M. G. & Schneider, M. F.(1999). Time course of individual voltage-activated Ca¥ sparksrecorded at ultra-high time resolution in frog skeletal muscle.Journal of General Physiology 113, 187—198.

Lamb, G. (2000). Excitation-contraction coupling in skeletal muscle.Comparison with cardiac muscle. Clinical and ExperimentalPharmacology and Physiology 27, 216—224.

Leong, P. & Maclennan, D. H. (1998). Complex interactions betweenskeletal muscle ryanodine receptor and dihydropyridine receptorproteins. Journal of Biochemistry and Cell Biology 76, 681—694.

Marx, S. O., Ondrias, K. & Marks, A. R. (1998). Coupled gatingbetween individual skeletal muscle Ca release channels (ryanodinereceptors). Science 281, 818—821.

Niggli, E. (1999). Localized intracellular calcium signaling in muscle:calcium sparks and calcium quarks. Annual Review of Physiology61, 311—335.

Parker, I., Zang, W. J. & Wier, W. G. (1996). Ca¥ sparks involvingmultiple Ca¥ release sites along Z-lines in rat heart cells. Journal ofPhysiology 497, 31—38.

R�úos, E., Stern, M. D., Gonz �alez, A., Pizarro, G. & Shirokova, N.(1999). Calcium release flux underlying Ca¥ sparks of frog skeletalmuscle. Journal of General Physiology 114, 31—48.

Schneider, M. F. (1999). Perspective: Ca¥ sparks in frog skeletalmuscle: generation by one, some, or many SR Ca¥ release channels?Journal of General Physiology 113, 365—372.

Sham, J. S., Song, L. S., Chen, Y., Deng, L. H., Stern, M. D.,Lakatta, E. G. & Cheng, H. (1998). Termination of Ca¥ release bya local inactivation of ryanodine receptors in cardiac myocytes.Proceedings of the National Academy of Sciences of the USA 95,15096—15101.

Shirokova., N., Gonzalez, A., Kirsch, W. G., Rios, E., Pizarro,G., Stern, M. D. & Cheng, H. (1999). Calcium sparks: releasepackets of uncertain origin and fundamental role. Journal ofGeneral Physiology 113, 377—384.

Shirokova, N. & R�úos, E. (1997). Small event Ca¥ release: a probableprecursor of Ca¥ sparks in frog skeletal muscle. Journal ofPhysiology 502, 3—11.

Simon, B. J., Klein, M. G. & Schneider, M. F. (1989). Caffeine slowsturn-off of calcium release in voltage clamped skeletal muscle fibers.Biophysical Journal 55, 793—797.

Stern, M. D., Pizarro, G. & R�úos, E. (1997). Local control model ofexcitation-contraction coupling in skeletal muscle. Journal ofGeneral Physiology 110, 415—440.

Tanaka, H., Nishimaru, K., Sekine, T., Kawanishi, T.,Nakamura, R., Yamagaki, K. & Shigenobu, K. (1997). Two-dimensional millisecond analysis of intracellular Ca¥ sparks incardiac myocytes by rapid scanning confocal microscopy: increasein amplitude by isoproterenol. Biochemical and Biophysical ResearchCommunications 233, 413—418.

Tsien, R. Y. (1990). Laser scanning confocal fluorescence microscopyat video rate (30 framesÏsec) with dual-wavelength emissionrationing for quantitative imaging of intracellular messages.Proceedings of the Royal Microscopical Society 25, S53.

Tsugorka, A., R� úos, E. & Blatter, L. A. (1995). Imagingelementary events of calcium release in skeletal muscle cells. Science269, 1723—1726.

Acknowledgements

We thank Dr Gonzalo Pizarro for stimulating discussions. This

project was supported by grants from National Institutes of Health

to E.R. (R01-AR32808) and N.S. (R01-AR45690).

Corresponding author

E. R� úos: Department of Molecular Biophysics and Physiology, Rush

University, 1750W. Harrison Street, Chicago, IL 60612, USA.

Email: [email protected]

Author’s present address

N. Shirokova: Department of Pharmacology and Physiology,

UMDNJ, New Jersey Medical School, Newark, NJ 07103, USA.




2000;528;419-433 J. Physiol.

Gustavo Brum, Adom González, Juliana Rengifo, Natalia Shirokova and Eduardo Ríos skeletal muscle

sparks in frog2+Fast imaging in two dimensions resolves extensive sources of Ca

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