Control of sarcoplasmic reticulum Ca2+ release by stochastic RyR gating within a 3D model of the cardiac dyad and importance of induction decay for CICR termination

Abstract

The factors responsible for the regulation of regenerative calcium-induced calcium release (CICR) during Ca(2+) spark evolution remain unclear. Cardiac ryanodine receptor (RyR) gating in rats and sheep was recorded at physiological Ca(2+), Mg(2+), and ATP levels and incorporated into a 3D model of the cardiac dyad, which reproduced the time course of Ca(2+) sparks, Ca(2+) blinks, and Ca(2+) spark restitution. The termination of CICR by induction decay in the model principally arose from the steep Ca(2+) dependence of RyR closed time, with the measured sarcoplasmic reticulum (SR) lumen Ca(2+) dependence of RyR gating making almost no contribution. The start of CICR termination was strongly dependent on the extent of local depletion of junctional SR Ca(2+), as well as the time course of local Ca(2+) gradients within the junctional space. Reducing the dimensions of the dyad junction reduced Ca(2+) spark amplitude by reducing the strength of regenerative feedback within CICR. A refractory period for Ca(2+) spark initiation and subsequent Ca(2+) spark amplitude restitution arose from 1), the extent to which the regenerative phase of CICR can be supported by the partially depleted junctional SR, and 2), the availability of releasable Ca(2+) in the junctional SR. The physical organization of RyRs within the junctional space had minimal effects on Ca(2+) spark amplitude when more than nine RyRs were present. Spark amplitude had a nonlinear dependence on RyR single-channel Ca(2+) flux, and was approximately halved by reducing the flux from 0.6 to 0.2 pA. Although rat and sheep RyRs had quite different Ca(2+) sensitivities, Ca(2+) spark amplitude was hardly affected. This suggests that moderate changes in RyR gating by second-messenger systems will principally alter the spatiotemporal properties of SR release, with smaller effects on the amount released.

Figure 1

Illustration of the model geometry. (A) Cross section of the cylindrical model with a t-tubule in the center. The j-SR wraps around the t-tubule at a 15-nm radial distance (dyad cleft). RyRs are located in the j-SR membrane and release Ca²⁺ into the dyad cleft. The j-SR contains calsequestrin (CSQ) and is connected to the n-SR by a tubule, which allows for Ca²⁺ diffusion between these two compartments. The SERCA Ca²⁺ uptake transporters are uniformly distributed throughout the n-SR. (B) Side view of the model in the cross section indicated by b-b in A. In this view, the t-tubule lumen runs horizontally and the j-SR can be seen on one side.

Figure 2

[Ca²⁺]-dependent RyR2 gating in lipid bilayers. Both sides of the bilayer contained (in mM) 230 CsCH₃O₃S and 20 CsCl (pH 7.4), with 2 ATP and 3 MgCl₂ on the cytoplasmic side ([Ca²⁺] as indicated). (A) Channel openings at various [Ca²⁺] are downward deflections from the baseline (arrows). (B) Cytoplasmic [Ca²⁺] dependence of P_o for RyRs from rat (luminal [Ca²⁺], 0.1 mM (solid circles) and 1 mM (open circles)) and sheep heart (luminal [Ca²⁺], 0.1 mM (open triangles)). Data points show the mean ± SE for n = 3–11 (solid circles), n = 4–8 (open circles), and n = 2–14 (open triangles)). Some of the rat data values have been replotted from Laver et al. (24). (C) Cytoplasmic [Ca²⁺] dependence of sheep RyR mean open and closed durations (0.1 mM luminal [Ca²⁺]). Solid symbols represent closed times and open symbols open times. Sheep RyR gating is shown by triangles and rat RyR gating, replotted from Laver et al. (24), by circles.

Figure 3

Simulation of Ca²⁺ sparks and blinks. (A) Simulated Ca²⁺ sparks. The simulated Ca²⁺ spark was generated by the array of RyRs shown in Fig. 4. (B) The corresponding simulated Ca²⁺ blink. Scale bars apply to both A and B. (C) Corresponding spatial profiles of spark and blink fluorescence at the times of their peak and minimum intensities, respectively. The noisy line shows the profile of an experimentally recorded Ca²⁺ spark (see also (23, 24) for additional comparison of model behavior to experimental data).

Figure 4

RyR cluster behavior during Ca²⁺ sparks. (A) Ca²⁺ spark and Ca²⁺ blink time courses for a cluster of 21 RyRs with a distribution as shown in D. (B) Spatial mean of [Ca²⁺] in the dyadic cleft and j-SR. (C) Numbers of open RyRs corresponding to the means in B. In A–C, line thickness shows the SD of 10 simulations. (D) Spatial distribution of [Ca²⁺] in the dyad at several time points during a typical simulation. RyRs are located at the circles, with solid and open circles representing open and closed RyRs, respectively. (E) Simulated time course of j-SR Ca²⁺ flux (wide trace) compared to the Ca²⁺ flux calculated from experimental Ca²⁺ spark profiles (line) replotted from Soeller and Cannell (43).

Figure 5

Effect of RyR Ca²⁺ sensitivity on Ca²⁺ spark activation and termination. (A) Mean time courses of Ca²⁺ spark and blink signals generated using a square cluster of 16 RyRs with Ca²⁺-activation kinetics derived from rat (n = 10) and sheep data (n = 3). (B) Corresponding spatial means of [Ca²⁺] in the dyadic cleft and j-SR. (C) Number of open RyRs for rat and sheep Ca²⁺-spark simulations, showing the mean (line) ± SD, (wide trace).

Figure 6

Intradyad geometric effects. (A) Ca²⁺ spark and blink time courses for two geometric arrangements of 18 RyRs. (Inset) Cluster organization with color key. (B) Evolution of mean P_o (shown as nP_o) for the two clusters indicated in A. (C) Spatial distribution of [Ca²⁺] in the dyad at several time points for the clusters shown in A. RyR states are depicted as described in Fig. 4 legend. (D) Effect of dyad dimensions. For a square cluster of 16 RyRs, the dyad space was set to the dimensions shown. Note that the smaller dyad results in faster Ca²⁺ spark termination but with a relatively small effect on amplitude.

Figure 7

Ca²⁺ spark restitution and i_RyR dependence. (A) Amplitude of the second Ca²⁺ spark (line) compared to experimental data (circles, replotted from Sobie et al. (25)). (B) Probability of triggering a second Ca²⁺ spark after various delays from the first. The trigger was assumed to be spontaneous RyR openings of 1 ms duration that occurred at intervals of 10 ms (left dashed line), 20 ms (solid line) and 40 ms (right dashed line). Data points (gray) are replotted from Sobie et al. (25). (C) Comparison of the time courses of j-SR [Ca²⁺] (dashed line), peak fluorescence (open circles) and nP_o (crosses). (D) Effect of i_RyR on Ca²⁺ spark amplitude. Sparks were simulated in a dyad containing 16 RyRs in a 4 × 4 array. The Ca²⁺ permeability of the RyR was varied and is expressed on the abscissa as Ca²⁺ current through the RyR in the presence of a 1 mM [Ca²⁺] difference across the SR membrane. Data show the mean ± SD for 6–17 simulations.