Sarcoplasmic reticulum Ca2+ release flux underlying Ca2+ sparks in cardiac muscle

Abstract

Discrete events of Ca2+ release from the sarcoplasmic reticulum (SR) have been described in cardiac, skeletal, and smooth muscle. In skeletal muscle these release events originate at individual channels. In cardiac muscle, however, it remains a question of debate whether localized Ca2+ release transients, termed Ca2+ sparks, originate from single release channels or multiple channels clustered in close vicinity. Generalizing methods used earlier to describe cell-averaged Ca2+ release, we derived, as a function of space and time, the flux of Ca2+ release that underlies Ca2+ sparks. Using the method to analyze spontaneous sparks recorded with confocal microscopy in dissociated cat atrial cells, we obtained in most cases single sparks of Ca2+ release that appear to originate from approximately 1-microm-wide regions. In many cases, doublets, triplets, and greater groups of release sparks were observed. This multiplicity, the estimated release flux magnitude, and existing data on the structure of junctions between SR and plasmalemma suggest that individual release sparks result from the opening of multiple Ca2+ release channels clustered within discrete SR junctional regions.

Figure 1

Repetitive Ca²⁺ sparks in cat atrial myocytes. (a) Confocal image (x–y scan; 512 × 512 pixels; pixel size = 0.17 μm) of fluo-3 fluorescence, obtained in a freshly isolated cell. (b) Line scan image obtained by repetitive scanning of the subsarcolemmal space every 4 ms along the line in a. The line scan image was constructed by stacking 512 lines vertically with time running from bottom to top as indicated by the arrow. (c) Three-dimensional representation of a fluorescence spark (fluorescence recorded at 8-bit resolution represented linearly by 256 levels of gray). The resting fluorescence, averaged during the initial 32 ms, varied with position around a value of 48, which was used to derive total dye concentration dye_T(x) (≃50 μM). The maximum of fluorescence was 180. (d) [Ca²⁺]_i(x, t), derived as described in Materials and Methods. Arrows calibrate time as listed.

Figure 2

The components of Ca²⁺ release flux. All terms in Eq. 5 were derived numerically from fluorescence and [Ca²⁺]_i records in Fig. 1. (a) Ca²⁺ diffusion term, −3D_Ca∂²[Ca²⁺]/∂x², with D_Ca = 3 × 10⁻⁶ cm²·s⁻¹ (20, 21). (b) Local rate of change of the Ca²⁺-bound indicator, ∂[Ca²⁺:dye]/∂t. (c) Flux of removal by a saturable diffusible buffer of 200 μM concentration, K_D = 0.4 μM, k_ON = 10⁸ M⁻¹·s⁻¹, and diffusion coefficient = 0.3 × 10⁻⁶ cm²/s. (d) Removal by the pump, with V_max = 1.4 mM/s, K_M = 0.3 μM, and transport rate proportional to the 2nd power of the occupancy (the two terms together constitute ∂rem/∂t in Eq. 5.) (e and f) Two representations of the release flux. In the color plot, blue = 0 and yellow = 10 mM/s. The line plots are averages of release over 40 ms (horizontal plot) or 1.2 μm (vertical) centered at the peak (dashed lines at 0). All time arrows span 50 ms; space calibrations, 3 μm; vertical bars calibrate flux (in mM/s) as indicated.

Figure 3

Multifocal release sparks. (a–e) Repeated fluorescence sparks, originated at the same spot in the scan, 13 μm from the edge of the cell. Shown are the first 5 of 36 wide sparks generated in a 10-min interval. (f) A narrower spark that originated at the same spot (no. 11 in the sequence). (g) Average fluorescence in the five sparks in a–e. The average resting fluorescence was 29, the peak 165, and dye_T(x) ≃ 31 μM. (h) [Ca²⁺]_i(x, t), derived from the averaged fluorescence. (i and j) Two representations of the Ca²⁺ release flux, calculated with the parameters given in Fig. 2. (k–o) Release flux images derived from the individual fluorescence sparks, showing in all five, indications of three release foci. (p) Release from the fluorescence in f.

Figure 4

Tests of the release algorithm. Panels in the first row represent fluorescence data. (a) Fluorescence of Fig. 1. (c) A fluorescence spark for which the analysis yielded a doublet of release sparks. (e) The average fluorescence of Fig. 3g. (b, d, and f) Images obtained by randomizing the phases in the Fourier transforms of a, c, and e (the phases were given random values, with adequate symmetry to preserve real inverse transforms; the procedure was repeated four times for each source image; one is shown for each). Second row, release flux calculated from corresponding images in the first row, showing a single release spark for a, multiplets for c and e, and no discernible sparks for the noise images (the numbers in each panel represent the maximum, X, in the color table, and apply to release calculated from signal and corresponding noise). Third row, release flux calculated for the sum of the original fluorescence plus its corresponding noise (the two corresponding panels in first row) in the same color scale. Fourth row, release flux from a, c, and e, respectively, assuming that dye_T(x) was constant and equal to its average.

Figure 5

Propagating release. (Left) Fluorescence. (a) F₀(x) ≃ 40; dye_T(x) ≃ 42 μM; maximum fluorescence = 163. (b) F₀(x) ≃ 43; dye_T(x) ≃ 45 μM; maximum fluorescence = 248. (Right) Release flux, calculated with the same parameters as in previous figures (numbers represent the maximum in the color scale).