Address correspondence to S.M. Baylor, Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6085. Fax: (215) 573-5851; E-mail: baylor{at}mail.med.upenn.edu
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ABSTRACT |
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Key Words: spark mass ryanodine receptors excitation-contraction coupling frog muscle
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INTRODUCTION |
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In frog skeletal muscle, voltage-activated Ca2+ sparks differ substantially in intact and cut fibers. For example, the average values of decay time constant, full duration at half maximum (FDHM),* full width at half maximum (FWHM), and spark mass are 1.5- to threefold larger in cut fibers than in intact fibers (Table VII of Hollingworth et al., 2001; see also Table II below). The largest difference is for mass.
The first part of this article describes some of the properties of spark mass, which is defined as the volume integral of F/F. These studies show that the amount of Ca2+ bound to fluo-3 is proportional to mass times the total concentration of fluo-3 ([fluo-3T]), with a proportionality constant that depends on [Ca2+]R. In an intact fiber simulation with [fluo-3T] = 100 µM and [Ca2+]R = 50 nM (the values that apply to intact fibers; Hollingworth et al., 2001
), fluo-3 captures approximately one-fourth of the Ca2+ released during a spark. Since mass in cut fibers is several times that in intact fibers, whereas [fluo-3T] and [Ca2+]R are similar, it seems likely that SR Ca2+ release is larger in cut fiber sparks or that fluo-3 is able to capture a larger fraction of the released Ca2+, perhaps because of reduced intrinsic Ca2+ buffering in cut fibers. Other factors, however, may contribute to the differences in spark properties, including the microscope point-spread function (PSF), the ionic composition of the myoplasmic solution, and the procedures used for spark analysis.
The second part of this article describes computer modeling that helps identify the factors that underlie the differences between intact and cut fiber sparks. The spark model of Baylor et al. (2002), which successfully simulates sparks in intact fibers, was modified to mimic the conditions encountered in the cut fiber experiments. The new simulations show that the source flux required for sparks in cut fibers is 310 times that in intact fibers; the exact factor depends on the concentrations of [Ca2+]R and the myoplasmic Ca2+ buffering proteins such as troponin. Such an increase in Ca2+ source flux could arise from an increase in Ca2+ flux through one RYR or an increase in the number of active RYRs per spark, or both. In either case, it seems clear that the gating of RYRs, or their apparent single channel Ca2+ flux, is different in frog cut fibersand, perhaps, in other disrupted preparationsthan in frog intact fibers.
Some of the results have appeared in abstract form (Baylor et al., 2003; Chandler et al., 2003
).
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MATERIALS AND METHODS |
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Simulation of Sparks in Intact Fibers
Calculations were made with spark model 2 of Baylor et al. (2002). In brief, the myoplasm is assumed to be isotropic, with its constituents distributed homogeneously in the resting state. For computational purposes, the myoplasmic volume is divided into 101 spherically symmetric compartments that are centered at the source of Ca2+ release and extend to 5 µm from the source. A spark occurs when a brief flux of Ca2+ enters the innermost compartment, a sphere of radius 25 nm. The model is used to calculate, for different times and radial distances from the source, the concentration of myoplasmic-free Ca2+, the concentrations of the Ca2+-free and Ca2+-bound forms of the major intrinsic myoplasmic Ca2+ buffers (troponin, ATP, parvalbumin, and the SR Ca2+ pump), and the concentrations of the Ca2+-free and Ca2+-bound forms of fluo-3.
The model considers four different forms of fluo-3: Fluo (Ca2+-free, protein-free fluo-3), PrFluo (Ca2+-free, protein-bound fluo-3), CaFluo (Ca2+-bound, protein-free fluo-3), and CaPrFluo (Ca2+-bound, protein-bound fluo-3). The total concentration of Ca2+-bound fluo-3, denoted by [Cafluo-3], is given by
![]() | (1) |
CaFluo and CaPrFluo are strongly fluorescent with the same relative intensity (Harkins et al., 1993), denoted by Fmax, whereas Fluo and PrFluo are weakly fluorescent. To allow for the fluorescence of Ca2+-free indicator, it is useful to introduce a derived fluo-3 concentration variable, [FFluo], defined by
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Fmin/Fmax and F'min/Fmax represent, respectively, the fluorescence intensities of Fluo and PrFluo divided by that of CaFluo or CaPrFluo; their values are 0.005 and 0.01, respectively (Harkins et al., 1993). According to Eq. 2, [FFluo] represents the concentration of CaFluo (or CaPrFluo) that has the same fluorescence as the mixture of CaFluo, CaPrFluo, Fluo, and PrFluo. The value of [FFluo]R is proportional to [fluo-3T]. The proportionality constant is equal to 0.0422 for [Ca2+]R = 50 nM, 0.0608 for [Ca2+]R = 80 nM, and 0.0728 for [Ca2+]R = 100 nM.
F/F is calculated by convolving
[FFluo]/[FFluo]R with the microscope PSF. In general,
denotes a change in a variable and subscript R denotes its resting value. The values of the FWHM of the PSF are 0.2 µm in x and y and 0.5 µm in z, the same as those measured in the confocal microscope used in the intact fiber experiments (Hollingworth et al., 2001
). This model with a Ca2+ source flux of 2.5 pA for 4.6 ms provides a good description of Ca2+ sparks in intact fibers (Baylor et al., 2002
).
Simulation of Sparks in Cut Fibers
The model described above for intact fibers was modified to simulate Ca2+ sparks in cut fibers. Table I
lists the differences between the intact and cut fiber simulation conditions (columns 2 and 3, respectively). The information for intact fibers was taken from Hollingworth et al. (2001). The information for cut fibers was taken from experiments in the Schneider laboratory. These experiments were selected for comparison because sparks in the Schneider laboratory and ours were analyzed with similar functions in space and time (Klein et al., 1997
; Lacampagne et al., 1999
; see below).
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Procedures for Spark Analysis in Intact Fibers
The analysis of an intact fiber spark, both experimental and simulated, followed procedures described in Hollingworth et al. (2001). Briefly, a 3 x 3 smoothed x-t image was formed from the original
F/F x-t image and an autodetection program was used to tentatively identify a spark as a contiguous region with peak
F/F
0.3. The unsmoothed
F/F image was then used to form a
F/F vs. t waveform as the average of the three time lines at x0 - 0.2 µm, x0, and x0 + 0.2 µm; x0 denotes the spatial center of the spark determined by the autodetection program. This waveform was least-squares fitted with Eq. 1 of Hollingworth et al. (2001)
, which is based on the corrected version of Eq. 2 of Lacampagne et al. (1999)
. This equation assumes that
F/F vs. t starts abruptly, rises exponentially toward a maximum value, then terminates abruptly and decays exponentially to a baseline offset. The fit determines the 0100% rise time, time of peak (denoted t2), peak amplitude, decay time constant, and FDHM. Then, a
F/F vs. x waveform was obtained from the unsmoothed
F/F image as an average of two line scans, just before and just after t2. This waveform was least-squares fitted with a Gaussian function with baseline offset (Eq. 2 of Hollingworth et al., 2001
; see also Klein et al., 1997
) to determine FWHM at time of peak
F/F. Spark mass at time of peak
F/F was estimated with Eq. 8 of Hollingworth et al. (2001)
:
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Sparks were excluded from the analysis if the fitted parameters did not satisfy the broad acceptance criteria described in Hollingworth et al. (2001). With the standard model for intact fiber sparks, these criteria exclude <1% of the simulated sparks.
Procedures for Spark Analysis in Cut Fibers
The analysis of a simulated cut fiber spark followed procedures described in Klein et al. (1997), in Lacampagne et al. (1999)
, and in a personal communication with Dr. M.F. Schneider. It started with the autodetection routine used for intact fibers. A possible spark, with an initial estimate of x0, was identified in the 3 x 3 smoothed image. An initial
F/F vs. t waveform was formed from the smoothed image as the average of the three time lines at x0 - 0.2 µm, x0, and x0 + 0.2 µm. The time of peak of this waveform was used as the initial estimate of t2. A
F/F vs. x waveform was then formed from the 3 x 3 smoothed image as the average of the three line scans at t2 - 2 ms, t2, and t2 + 2 ms and was fitted with a gaussian function (Eq. 2 of Hollingworth et al., 2001
) to determine FWHM and the final estimate of x0. Finally, a
F/F vs. t waveform was obtained from the unsmoothed x-t image as an average of seven time lines at x0, x0 ± 0.2 µm, x0 ± 0.4 µm, and x0 ± 0.6 µm. This waveform was fitted with Eq. 1 of Hollingworth et al. (2001)
to determine 0100% rise time, peak amplitude, decay time constant, and FDHM. Analyzed sparks were accepted if peak amplitude satisfied
F/F
0.4 (Lacampagne et al., 1999
) and the other morphological parameters satisfied the broad acceptance criteria described in Hollingworth et al. (2001)
.
Spark Mass and its Equivalence to the Volume Integral of [FFluo]/[FFluo]R
F/F is given by the convolution of
[FFluo]/[FFluo]R with the microscope PSF,
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By changing the order of integration with respect to x', y', z' and x, y, z, and using the fact that the volume integral of PSF equals 1, M can be written
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Eq. 6 shows that M is equal to the increase in the total normalized amount of FFluo and that this equality does not depend on the spatial resolution of the confocal microscope. The equality holds for any PSF that is continuous in x, y, and z. Because the absolute value of [CaFluo] +
[CaPrFluo] is much greater than the absolute value of 0.005 ·
[Fluo] + 0.01 ·
[PrFluo],
[FFluo] is approximately equal to
[Cafluo-3], and
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Eq. 7 shows that the total amount of Ca2+ captured by fluo-3 is approximately equal to M(t) · [FFluo]R.
Statistics
For each set of noisy-spark simulations in Tables IV, V, and VII, sufficient sparks were generated to give 3,176 sparks for inclusion in the analysis. This number is the same as that in the measurements of Hollingworth et al. (2001) and in the simulations of Baylor et al. (2002)
. Values of the morphological parameters are reported as mean ± SEM. The statistical significance of a difference between means was evaluated with Student's two-tailed t test at P < 0.05.
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RESULTS |
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Spark Mass Equals the Volume Integral of [FFluo]/[FFluo]R
Fig. 1
A shows the time course of F/F at the Ca2+ source for a standard noise-free simulated spark. The peak amplitude is 2.14 and the time of peak is 4.6 ms, the same as the flux duration. Fig. 1 B shows two nearly identical curves. One is the time course of "true" mass, M(t), calculated from its definition (Eq. 5). The other is the time course of the volume integral of
[FFluo]/[FFluo]R, which is equal to spark mass (Eq. 6); this equality does not depend on the spatial distribution of
[FFluo]/[FFluo]R or on the microscope PSF (see MATERIALS AND METHODS). As expected from the theory, the two curves in Fig. 1 B are indistinguishable. At the time of peak
F/F (4.6 ms), the value of mass is 2.64 µm3. Although the Ca2+ source flux ceases at 4.6 ms, M(t) continues to increase; it reaches its peak value, 3.63 µm3, at 10.8 ms, 6.2 ms after the peak
F/F. The lag between cessation of Ca2+ release and the peak of mass arises from kinetic delays in the reactions between Ca2+ and fluo-3 in the myoplasmic environment (Harkins et al., 1993
; Baylor and Hollingworth, 1998
; Hollingworth et al., 2000
). After 10.8 ms, mass decreases as Ca2+ dissociates from fluo-3 and is captured by parvalbumin and the SR Ca2+ pump.
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The dashed curve in Fig. 1 C shows M(t). According to Eq. 7, which is illustrated by the similarity of the dashed and continuous curves in Fig. 1 C, the volume integral of [Cafluo-3] is expected to be approximately equal to M(t) times [FFluo]R. The peak value of M(t) (3.63 µm3) times [FFluo]R (4.22 µM) gives 9,236 for the number of Ca2+ ions captured by fluo-3, which is 0.99 times the actual value.
Use of Eq. 3 to Estimate Spark Mass
Although spark mass depends on the spatial spread of F/F in three dimensions, its value can be estimated with Eq. 3 from the spatial spread in the x direction only. Fig. 2
shows noise-free calculations that illustrate the estimation of spark mass (Me). Fig. 2, A and B, shows the time courses of
F/F and FWHM, respectively, at the source; these were obtained from fits of a gaussian function to the waveform of
F/F vs. x at different times t. The curve in Fig. 2 A differs slightly from that in Fig. 1 A, which is the actual temporal waveform of
F/F at the source. This difference arises because
F/F vs. x is not an exact gaussian function, either in the simulations or in the measurements (Fig. 9, B and E, of Baylor et al., 2002
). In spite of this, the
F/F vs. t waveforms in Figs. 1 A and 2 A have similar peak amplitudes, the same time of peak (4.6 ms, which is the time at which the Ca2+ source flux terminates), and very similar overall time courses.
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Time Course of Mass in Simulated Noisy Sparks and in Sparks in Intact Fibers
Fig. 3
shows simulated data (asterisks) and measured data (open squares); 0 ms denotes the estimated time of peak F/F. Each set of data was obtained from an average of 179 in-focus sparks, defined as the largest 10% of sparks with peak amplitude
F/F
0.7. Noise and variability were included in the simulated data to mimic the measurements (see MATERIALS AND METHODS). Fig. 3 also shows the continuous curves from Fig. 2 time-shifted by -4.6 ms so that 0 ms corresponds to the time of peak
F/F. Fig. 3, A and B, show the time course of
F/F and Fig. 3, C and D, show FWHM. Both the simulated and measured values of FWHM become noisy after 12 ms; this occurs because
F/F becomes small and the noise in
F/F vs. x makes the gaussian fits less reliable. The simulated data in these panels are in reasonable agreement with the measured data, and, within the noise, both sets of data lie close to the curves, at least out to
40 ms.
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The simulations and measurements in Figs. 13 indicate that Eq. 3 provides reasonable estimates of the peak mass and time of peak mass of an in-focus spark.
Dependence of Simulated Spark Mass on the Amount of Ca2+ Released
Noise-free simulations of sparks at the source of Ca2+ flux were also used to study the dependence of M and Me on the total amount of SR Ca2+ released during a spark. Ca2+ release was varied by changing either the amplitude or the duration of the source flux. Fig. 4
shows mass at the time of peak F/F (A) and at the time of peak true mass (B) plotted against the amount of Ca2+ released. For releases up to
30 fC, both true mass (filled symbols) and estimated mass (open symbols) vary approximately linearly with the amount of Ca2+ released. In both panels, the slope of the line fitted to estimated mass (dashed line) is smaller than that fitted to true mass (continuous line). The ratio of the slopes (dashed divided by continuous) is 0.545 in A and 0.832 in B. These simulations show that, for the range of Ca2+ releases considered, both the true and estimated mass of an in-focus spark are approximately proportional to the amount of SR Ca2+ released, with a proportionality constant that is smaller at the time of peak
F/F than at the time of peak mass. The proportionality constant for peak true mass (slope of the continuous line in Fig. 4 B) corresponds to the capture of 24.9% of the Ca2+ released from the SR by fluo-3 ([fluo-3T] = 100 µM).
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Trace e was obtained in the same manner as trace d except that cut fiber conditions were used for the simulations (Table I A, column 3). The smaller amplitude of trace e is due mainly to the increase in [Ca]R from 50 to 80 nM. This increases the resting concentration of Ca2+-bound fluo-3 and hence resting fluorescence; as a result, a smaller F/F signal is produced for a given Ca2+ flux (e.g., Jiang et al., 1999
; Baylor et al., 2002
).
The peak F/F amplitudes in Fig. 5 A progressively decrease from a to e. Trace b, with a peak value of 1.808, represents the temporal waveform of a noise-free simulated intact fiber spark with the scan line through the Ca2+ source. Trace e, with a peak value of 0.522, is the comparable waveform for a cut fiber spark. These simulations show that, with a Ca2+ source flux of 2.5 pA for 4.6 ms and with the line scan through the Ca2+ source, a spark measured in a cut fiber is expected to have an amplitude that is
0.3 times that in an intact fiber.
Fig. 5 B shows the spatial waveforms of F/F that accompany the traces in A. All waveforms in B have been scaled to a peak amplitude of unity to facilitate the comparison of the spatial spread of the sparks. The FWHMs of the waveforms progressively increase from 0.740 µm in a to 1.177 µm in d; waveforms in d and e are indistinguishable.
Additional information about the simulations in Fig. 5 is given in Table III , columns 26. From the intact fiber simulation of column 3 to the cut fiber simulation of column 6, there is a 71% reduction in peak amplitude, an 18% increase in FDHM, a 39% increase in FWHM, and a 23% reduction in spark mass.
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Simulations with Reduced [Ca2+]R (Ca2+ Source Flux = 2.5 pA x 4.6 ms)
Although the value of [Ca2+]R in cut fibers appears to be larger than that in intact fibers (Hollingworth et al., 2001; see also DISCUSSION), it was nonetheless of interest to determine the effect of reducing [Ca2+]R from 80 to 50 nM, the standard value used for spark simulations in intact fibers (Table I). This reduction is expected to reduce resting F and therefore increase
F/F for a given Ca2+ flux. Table IV, columns 57, are similar to columns 24 except that [Ca2+]R = 50 nM. Even without troponin (column 7), the mean values of spark amplitude and mass (0.630 and 2.368 µm3, respectively) are substantially smaller than those of the measurements (1.05 and 4.36 µm3, respectively).
Our conclusion from the results in Table IV is that a Ca2+ flux of 2.5 pA is too small to account for the amplitude and some of the other properties of sparks in cut fibers.
Simulation of Noisy Sparks in Cut Fibers with Mean F/F
1.05 (Ca2+ Source Flux > 2.5 pA for 4.6 ms)
Table V
shows results similar to those in Table IV except that, for each simulation condition, the Ca2+ flux amplitude was increased in units of 1 pA until average F/F was
1.05, similar to that of the cut fiber measurements. In these simulations, sparks that satisfy the criterion
F/F
0.4 can be detected farther from the source so that the average values of D in Table V are substantially larger than those in Table IV. The values of the other parameters in Table V, columns 27, are broadly consistent with the experimental results in Table II, column 3. Consequently, none of the six combinations of [troponin] and [Ca2+]R can be definitely ruled out. As expected, the largest Ca2+ flux (23 pA, column 2) occurs with the standard values of [troponin] and [Ca2+]R, and the smallest flux (8 pA, column 7) occurs with [troponin] = 0 and [Ca2+]R = 50 nM. Even the 8 pA value is more than three times that required for the simulation of sparks in intact fibers, 2.5 pA.
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Simulations with Increased Myoplasmic Diffusion Constants and Increased Myoplasmic Water Volume
Table VI
shows the apparent diffusion constants of six indicator dyes studied in cut fibers in the Chandler laboratory and in intact fibers in the Baylor laboratory. On average, apparent diffusion constants in cut fibers are 1.3 times those in intact fibers (Table VI, column 4). A possible explanation, which is supported by the measurements of Irving et al. (1987)
, is that the myoplasmic water volume is increased in cut fibers compared with intact fibers. These authors measured intrinsic birefringence (optical retardation per unit path length, which primarily reflects the birefringence of myosin) in both intact and cut fibers and found that cut fibers, on average, have values that are
0.85 times those in intact fibers. This suggests that the optical path length in cut fibers is 1/0.85 times that in intact fibers, and that myoplasmic water volume is increased according to the factor 1.4 (
1/0.852). An increase in water volume would be expected to reduce the viscosity of myoplasm and, thus, to increase the actual diffusion constants of all diffusible myoplasmic constituents (including the indicator dyes). An increase in water volume would also be expected to dilute the concentrations of poorly diffusible myoplasmic constituents of high molecular weight, such as soluble and structural proteins, to which indicator molecules readily bind (e.g., Konishi et al., 1988
; Kurebayashi et al., 1993
). This reduction in concentration of binding sites would be expected to further increase the apparent diffusion constants of the indicators.
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DISCUSSION |
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Signal Mass in Nonmuscle Cells
The concept of signal mass was introduced by Sun et al. (1998), who studied Ca2+ signaling events ("blips" and "puffs") mediated by inositol-tris-phosphate in oocytes injected with the fluorescent Ca2+ indicator Oregon green 488 Bapta-1. Signal mass (the volume integral of
F/F) was estimated from
F/F vs. x with a method that is different from that used in this article. During blips (the smallest resolved events,
F/F
0.25), signal mass increased at about the same time as
F/F or shortly thereafter, and peak mass (
5 µm3) was reached
15 ms after peak
F/F (Fig. 4 C of Sun et al., 1998
). During puffs (larger events,
F/F
12), the peak value of mass was an order of magnitude larger (
80 µm3) and it occurred
100 ms after the peak of
F/F (Fig. 4 D of Sun et al., 1998
). The delay from peak
F/F to peak mass was attributed to a continued but diminished flux of Ca2+ into the cytoplasm.
Thus, the peak values of mass in oocytes are either comparable to, or many times larger than, that estimated for an averaged in-focus spark in frog intact muscle fibers (5100 µm3 compared with 3.8 µm3) and the lag between peak
F/F and peak mass in oocytes is at least several times larger than that in intact muscle fibers (
15 ms and
100 ms compared with
6 ms). Some of the lag in oocytes is likely due to kinetic delays in the reactions between Ca2+ and the indicator in the cytoplasmic environment, similar to the situation with sparks in frog intact muscle fibers.
Signal Mass in Cut Fibers
As far as we are aware, the only estimate of mass in cut muscle fibers was reported by Gonzalez et al. (2000). With Eq. 3, however, mass can be calculated from other articles if the values of
F/F and FWHM are given. Table VII in Hollingworth et al. (2001)
tabulates such values at the time of peak
F/F: 3.75.2 µm3 for voltage-activated sparks in cut fibers and 4.422.5 µm3 for spontaneous sparks in permeabilized cut fibers (amplitude threshold for spark acceptance,
F/F
0.5 to 1.0). These values of mass are 2.515 times those obtained for voltage-activated sparks in intact fibers at the time of peak
F/F, 1.41.5 µm3.
In the paper by Gonzalez et al. (2000), frog fibers were permeabilized by saponin and exposed to Imperatoxin A, an agent that, in bilayer experiments, binds to open RYRs and induces a long-lived substate that has about one-third the normal conductance (Tripathy et al., 1998
). The toxin-related events usually had an initial
F/F that was similar to a spark followed by a small maintained
F/F that lasted
1 s (Gonzalez et al., 2000
). The spark-like event in their Fig. 2, AD, had a peak
F/F
3, a FWHM at time of peak
F/F
1.9 µm, and a peak mass
50 µm3. An average of nine such events was simulated with a Ca2+ source flux of peak amplitude of
11 pA and half-duration of
9 ms (Fig. 2 F of Gonzalez et al., 2000
). Both the peak mass of the single event, 50 µm3, and the amount of Ca2+ release estimated for the averaged event,
99 fC, are an order of magnitude larger than the values estimated for in-focus sparks activated by voltage in frog intact fibers, 34 µm3 (Fig. 3, E and F) and 11.5 fC, respectively.
Simulation of Sparks in Cut Fibers
The main conclusion of this article is that the simulation of Ca2+ sparks in cut fibers requires a Ca2+ source flux that is substantially larger than the 2.5 pA required to simulate sparks in intact fibers. The required source flux is also substantially larger than the 1.4 pA used in the spark simulations by the Schneider laboratory (Jiang et al., 1999). With the standard concentrations of troponin and resting Ca2+ in the cut fiber model, a Ca2+ source flux of 23 pA is required (Table V, column 2). Even under the extreme conditions that [Ca2+]R = 50 nM and the troponin regulatory sites bind no Ca2+ at all, a source flux of 8 pA is required (Table V, column 7), which is three times that required in intact fibers. Because the values of the morphological parameters in each row of Table V, columns 27, are close to one another and to those in the cut fiber experiments (Table II, column 3), all six model conditions in Table V produce a reasonable simulation of sparks in cut fibers. Thus, these simulations do not establish the likely value of [Ca2+]R or the concentration of the troponin sites available for Ca2+ binding. Similar conclusions apply to the simulations in Table VII, which include increases in myoplasmic diffusion constants and myoplasmic water volume. These simulations, which are in less satisfactory agreement with the measurements than those in Table V, also required large Ca2+ source fluxes (922 pA).
Comparisons with the Measurements and Simulations by the Ríos Laboratory
Voltage-activated Ca2+ sparks in cut fibers appear to be different in the Ríos and Schneider laboratories (Table VII of Hollingworth et al., 2001). For example, spark amplitude is substantially larger in the Ríos laboratory (1.85 ± 0.12; amplitude acceptance criterion,
F/F
0.6; 18 ± 1°C), even though the values of FWHM for the microscope PSF in the Ríos laboratory (0.47 µm in x and y and 1.44 µm in z; Ríos et al., 1999
) are similar to or larger than those in the Schneider laboratory (0.5 µm in x and y and 1.0 µm in z, respectively; Table I, column 3). Since FWHM is slightly smaller in the Ríos laboratory (1.33 vs. 1.5 µm; Table VII of Hollingworth et al., 2001
), spark mass is only slightly larger (5.2 vs. 4.36 µm3). The larger spark amplitude and slightly larger value of mass in the Ríos laboratory make it likely that the underlying Ca2+ source flux is at least as large as the 823 pA required for the simulation of sparks from the Schneider laboratory (row 3 of Tables V and VII). This expectation is in agreement with spark simulations by the Ríos laboratory, which required Ca2+ source fluxes of 8 to 27 pA, depending on conditions (Table IV of Ríos et al., 1999
).
Significance of a Larger Amplitude Ca2+ Source Flux in Cut Fibers
A larger Ca2+ source flux in cut fibers could be caused by an increase in RYR single channel Ca2+ flux, an increase in mean open probability, an increase in the number of active RYRs per spark, or a combination of these possibilities. There are several differences between cut and intact fibers that might explain such an increase(s). First, as considered in the last section of RESULTS, cut fibers appear to be more hydrated than intact fibers and this swelling might alter RYR function, perhaps by changing the physical interactions between adjacent RYRs or between the RYRs and other proteins at the triadic junction. Second, the relative amplitude of fluo-3's resting fluorescence at the z- and m-lines differs between intact fibers (Hollingworth et al., 2001) and cut fibers (Tsugorka et al., 1995
; Klein et al., 1996
; Lacampagne et al., 1996
; Shirokova and Ríos, 1997
). The cut fiber pattern can be mimicked in intact fibers by increasing the concentration of K+ in the bathing solution from 2.5 to 7.530 mM. Since an increase in [Ca2+]R accompanies an elevation in [K+], it seems likely that the pattern of fluo-3's resting fluorescence is a rough indicator of [Ca2+]R. By this criterion, [Ca2+]R is larger in cut fibers than in intact fibers (Hollingworth et al., 2001
). Third, the duration of an action-potentialstimulated Ca2+ transient progressively increases with time during a 2 h experiment in a cut, but not an intact, fiber (Maylie et al., 1987b
,c
). This increase, which occurs in the absence of changes in indicator concentration, suggests that Ca2+ uptake is progressively slowed during the 2-h period, perhaps because of a progressive loss of intrinsic myoplasmic Ca2+ buffers or of modulators that maintain the normal activity of the SR Ca2+ pump; a decrease in the concentration of parvalbumin does not appear to occur during this period (Irving et al., 1989
). In addition to these three documented differences between cut and intact fibers, small mobile molecules such as monovalent and divalent ions, ATP, phosphocreatine, and peptides would be expected to diffuse out of a fiber after cutting so that the composition of myoplasm in cut fibers would be expected to become progressively different from that in intact fibers (even though additions are usually made to the cut fiber end-pool solution to keep the concentrations of some of these constituents near the normal range). For example, [Mg2+]R and [Ca2+]R, which strongly affect RYR function, are probably 0.50.7 mM and 0.080.1 µM, respectively, in cut fibers and
1 mM and
0.05 µM, respectively, in intact fibers (Table I).
The differences between cut and intact fibers listed above might account for some, perhaps all, of the increased Ca2+ source flux in cut fiber sparks. For example, an increase in [Ca2+]R in cut fibers would be expected to increase the activity of the SR Ca2+ pump, which, in turn, should increase free [Ca2+] inside the SR and thereby increase RYR single channel Ca2+ flux. The diffusive loss of small molecules from the myoplasm of cut fibers could, in theory, increase the Ca2+ flux through an RYR if channel blockers or modulators that decrease the mean open probability were removed. The smaller value of [Mg2+]R and the larger value of [Ca2+]R (and the possible associated increase in SR Ca2+ content) in cut fibers could increase the number of RYRs per spark by augmenting Ca2+-induced Ca2+ release, a process that has been described in cut fibers (Jacquemond et al., 1991; Stern et al., 1997
; Gonzalez et al., 2000
; see also Ríos and Pizarro, 1988
). Although the cause(s) of the increased Ca2+ source flux in sparks in cut fibers is poorly understood at this time, the presence of this difference between RYR function in intact and cut fibers suggests that intact fibers contain structural or regulatory factors that are altered or missing in cut fibersand, perhaps, in other disrupted preparations.
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FOOTNOTES |
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ACKNOWLEDGMENTS |
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This work was supported by grants from the U.S. National Institutes of Health to W.K. Chandler (AM 37643) and S.M. Baylor (NS 17620).
Olaf S. Andersen served as editor.
Submitted: 3 January 2003
Revised: 27 February 2003
Accepted: 28 February 2003
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REFERENCES |
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