Ca2+ spark latency and control of intrinsic Ca2+ release dyssynchrony in rat cardiac ventricular muscle cells

Abstract

Cardiac excitation-contraction coupling (ECC) depends on Ca²⁺ release from intracellular stores via ryanodine receptors (RyRs) triggered by L-type Ca²⁺ channels (LCCs). Uncertain numbers of RyRs and LCCs form 'couplons' whose activation produces Ca²⁺ sparks, which summate to form a cell-wide Ca²⁺ transient that switches on contraction. Voltage (V_m) changes during the action potential (AP) and stochasticity in channel gating should create variability in Ca²⁺ spark timing, but Ca²⁺ transient wavefronts have remarkable uniformity. To examine how this is achieved, we measured the V_m-dependence of evoked Ca²⁺ spark probability (P_spark) and latency over a wide voltage range in rat ventricular cells. With depolarising steps, Ca²⁺ spark latency showed a U-shaped V_m-dependence, while repolarising steps from 50 mV produced Ca²⁺ spark latencies that increased monotonically with V_m. A computer model based on reported channel gating and geometry reproduced our experimental data and revealed a likely RyR:LCC stoichiometry of ∼ 5:1 for the Ca²⁺ spark initiating complex (IC). Using the experimental AP waveform, the model revealed a high coupling fidelity (P_cpl ∼ 0.5) between each LCC opening and IC activation. The presence of ∼ 4 ICs per couplon reduced Ca²⁺ spark latency and increased P_spark to match experimental data. Variability in AP release timing is less than that seen with voltage steps because the AP overshoot and later repolarization decrease P_spark due to effects on LCC flux and LCC deactivation respectively. This work provides a framework for explaining the V_m- and time-dependence of P_spark, and indicates how ion channel dispersion in disease can contribute to dyssynchrony in Ca²⁺ release.

Keywords: Action potential; Ca(2+) sparks; Cardiac myocytes; Excitation-contraction coupling; Heart; Intracellular Ca(2+).

Fig. 1. Ca²⁺ release latency during an AP.

(A) Top panel shows the AP voltage-clamp waveform and the bottom panel the evoked Ca²⁺ current (nifedipine-sensitive). The insets show the same data at a slower time scale. Amplifier saturation effects during the upstroke of the AP were not completely removed by subtraction of the current recorded in nifedipine and led to an artifact at early times as indicated in grey. (B) Confocal line scan image of the normalized Fluo-5F fluorescence (F/F₀) changes evoked by the AP shown in A. The transmitted light intensity (TL) of the line scan region is shown at the left and indicates z-line positions. The time base is the same for panels A & B. Note the high time resolution and relative uniformity in Ca²⁺ release at z-lines. The bottom panel shows the mean time course of Fluo-5F fluorescence (black line) together with local time course at the positions marked by the arrow heads. (C) Ca²⁺ sparks evoked by sequential AP clamp pulses in the presence of nifedipine. Note the much lower probability of Ca²⁺ release along the scan line and the beat-to-beat variation in position and timing. The red arrows indicate when V_m exceeds −40 mV during the AP upstroke. (D) Ca²⁺ spark (red bars, 170 events, n/N = 6/3) and Ca²⁺ transient latencies (blue bars, 721 z-lines, n/N = 7/3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Ca²⁺ spark latencies in response to DP and RP.

(A) Exemplar confocal line scan images showing Ca²⁺ sparks in the presence of nifedipine at the indicated DP V_m. (B) Ca²⁺ spark rate during the first 20 ms (838 Ca²⁺ sparks, n/N = 14/6) (filled circles). The maximum rate of rise for Ca²⁺ transients for the same DP Vm is shown by blue triangles (218 Ca²⁺ transients, n/N = 14/8). There was no difference between these V_m dependencies when normalized (p > 0.999, Chi-squared test). (C) Corresponding V_m-dependence of mean (solid circles), median and interquartile range (grey bars) Ca²⁺ spark latency (1215 Ca²⁺ sparks, n/N = 14/8). The solid blue line shows an empirical bi-exponential fit to the mean data:${\text{Latency}}_{\text{DP}}=6.7\left({\text{e}}^{-\frac{\text{v}}{18.5}}+{\text{e}}^{\frac{\text{v}}{20.8}}\right)$ (r² = 0.99). (D) Ca²⁺ sparks in response to RP (same cell as A). The inset shows the distribution of Ca²⁺ spark latency during DP to −30 mV. (E) V_m-dependence of Ca²⁺ spark latency during RP in the presence of nifedipine (1201 Ca²⁺ sparks, n/N = 15/7). The solid blue line shows an empirical exponential fit: ${\text{Latency}}_{\text{RP}}=9.2{\text{e}}^{\frac{\text{v}}{39.6}}+3.3$ (r² = 0.96). The inset shows the distribution of Ca²⁺ spark latency during RP to −30 mV. (F) The coefficient of variation for DP-, RP- and AP-evoked Ca²⁺ spark latencies. For DP and RP, the V_m were − 30 to 10 mV (p-values from Mann-Whitney tests). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Simulation of ECC using reported LCC, RyR and dyad properties.

(A) Time course of average LCC Ca²⁺ flux by DP to the V_m indicated (5000 simulations each). (B) V_m-dependence of peak LCC Ca²⁺ flux from A. Note that the averaged single LCC currents in A,B are very similar to reported whole cell I_Ca data (e.g. [24,34]). (C) and (D) show experimental (black symbols, from Josephson et al. [27]) and model (red lines) for P_O,LCC and k_off,LCC respectively (E) A distance map for RyRs with RyR positions as shown by Asghari et al. [41]. (F) Calculated [Ca²⁺]_dyad 4 nm from the surface sarcolemma at the indicated lateral distances from an open LCC (i_Ca = 0.2 pA for 1 ms). The lateral distances shown were chosen so that these data can be directly compared Soeller & Cannell [30], as well as to the RyR distance map shown in panel E. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Ca²⁺ release latency during voltage-clamp steps.

(A) Diagram showing modelled processes that contribute to the calculated Ca²⁺ release latency (from top to bottom): (i) DP to 0 mV with finite clamp speed (τ ~ 0.3 ms), (ii) LCC transitions from closed (‘C’) to open (‘O’) states, (iii) the corresponding Ca²⁺ influx (i_Ca), (iv) [Ca²⁺]_dyad at an RyR location (indicated by colour, see inset). RyR locations were derived from electron microscopy data [41]. (v) Net RyR on rate and (vi) cumulative RyR open probability (∑p_{o, RyR}), where the first opening that triggers Ca²⁺ release is indicated (red arrow). (B) Simulated mean (solid lines) and 95% confidence intervals for Ca²⁺ release latencies for various RyR:LCC ratios and RP = ‒15, 0 and 15 mV (4000 simulations for each data point). The measured latencies during RP are shown at left (from Fig. 2E). (C) Simulated V_m-dependence of Ca²⁺ release latency for a 4:1 (purple) or 8:1 (green) RyR:LCC stoichiometry. (D) Corresponding Ca²⁺ spark rate. Inset shows RyR arrangements for the simulations. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. P_spark and P_cpl during RP- and AP-evoked Ca²⁺ release.

(A) The fraction of Ca²⁺ sparks (F_spark) that were evoked by n_o LCC openings at each V_m (40 00 simulations each). The right-hand side shows the corresponding F_spark using the AP shown in Fig. 1A. The mean n_O was 2.01 ± 0.01 (10,000 simulations). (B) The V_m-dependence of coupling fidelity (P_cpl) during RP. P_cpl for an AP is shown by a cross on the right. C) The exemplar AP waveform used in the computer model. D) Simulated P_spark using a 4:1 RyR:LCC stoichiometry for 1 (red bars) or 4 (blue bars) ICs. The mean latencies were 9.55 ± 0.06 ms (P_spark = 0.796, 10,000 simulations) and 5.61 ± 0.02 ms (P_spark = 0.997, 10,000 simulations) respectively. E) Average i_Ca during the AP and proportion involved in triggering Ca²⁺ transients (blue region). F) The PDFs for Ca²⁺ spark initiation during an AP (red) or RP to 0 mV (black). The results in E and F are the average of 50,000 simulations and the origin of the reduction Ca²⁺ release dyssynchrony associated with the AP compared to RP is highlighted by the green shaded regions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)