Ca²+ spark-dependent and -independent sarcoplasmic reticulum Ca²+ leak in normal and failing rabbit ventricular myocytes

Abstract

Sarcoplasmic reticulum (SR) Ca²(+) leak is an important component of cardiac Ca²(+) signalling. Together with the SR Ca²(+)-ATPase (SERCA)-mediated Ca²(+) uptake, diastolic Ca²(+) leak determines SR Ca²(+) load and, therefore, the amplitude of Ca²(+) transients that initiate contraction. Spontaneous Ca²(+) sparks are thought to play a major role in SR Ca²(+) leak. In this study, we determined the quantitative contribution of sparks to SR Ca²(+) leak and tested the hypothesis that non-spark mediated Ca²(+) release also contributes to SR Ca²(+) leak. We simultaneously measured spark properties and intra-SR free Ca²(+) ([Ca²(+)](SR)) after complete inhibition of SERCA with thapsigargin in permeabilized rabbit ventricular myocytes. When [Ca²(+)](SR) declined to 279 ± 10 μm, spark activity ceased completely; however SR Ca²(+) leak continued, albeit at a slower rate. Analysis of sparks and [Ca²(+)](SR) revealed, that SR Ca²(+) leak increased as a function of [Ca²(+)](SR), with a particularly steep increase at higher [Ca²(+)](SR) ( >600 μm) where sparks become a major pathway of SR Ca²(+) leak. At low [Ca²(+)](SR) (< 300 μm), however, Ca²(+) leak occurred mostly as non-spark-mediated leak. Sensitization of ryanodine receptors (RyRs) with low doses of caffeine increased spark frequency and SR Ca²(+) leak. Complete inhibition of RyR abolished sparks and significantly decreased SR Ca²(+) leak, but did not prevent it entirely, suggesting the existence of RyR-independent Ca²(+) leak. Finally, we found that RyR-mediated Ca²(+) leak was enhanced in myocytes from failing rabbit hearts. These results show that RyRs are the main, but not sole contributor to SR Ca²(+) leak. RyR-mediated leak occurs in part as Ca²(+) sparks, but there is clearly RyR-mediated but Ca²(+) sparks independent leak.

Figure 4. Effects of RyR stimulation by low-dose caffeine on Ca²⁺ sparks, [Ca²⁺]_SR and SR Ca²⁺ leak

A, line-scan images and corresponding profiles of Rhod-2 (red) and Fluo-5N (green) fluorescence in control conditions, in the presence of caffeine (200 μm) and after subsequent application of thapsigargin (TG; 10 μm). Fluo-5N was recorded with open pinhole (non-confocal setting) whereas Rhod-2 was recorded confocally. B, effect of caffeine (200 μm) followed by SERCA inhibition (10 μm TG) on spark frequency and [Ca²⁺]_SR. Application of 10 mm caffeine at the end of the experiment indicates complete depletion of the SR. Measurements were made from the same cell shown in panel A. C, average spark frequency and [Ca²⁺]_SR in control conditions, immediately (initial) and 2 min (late) after exposure to caffeine (200 μm). D, the relationships between SR Ca²⁺ leak rate and [Ca²⁺]_SR in control conditions (back) and in the presence of caffeine (red).

Figure 1. Simultaneous measurements of Ca²⁺ sparks and [Ca²⁺]_SR in permeabilized ventricular myocytes

A, line-scan images and corresponding profiles (F/F₀) of Rhod-2 (red) and Fluo-5N (green) fluorescence in control conditions and at different times after application of thapsigargin (TG; 10 μm). Fluo-5N was recorded with open pinhole (non-confocal setting) whereas Rhod-2 was recorded confocally. The Ca²⁺ spark profiles were obtained by averaging fluorescence from the 1 μm wide region marked by the red box. Fluo-5N profiles were obtained by averaging fluorescence over the entire width of the line-scan images. At the end of the experiment, F_min and F_max were measured (see Methods). B, relationship between initial [Ca²⁺]_SR and Ca²⁺ spark frequency measured under control conditions (before TG application) from 16 different cells. Frequency correlated positively with [Ca²⁺]_SR measured under control conditions (R²= 0.78). C, effects of SERCA inhibition on spark frequency and [Ca²⁺]_SR. Measurements were made from the same cell shown in panel A.

Figure 3. Contribution of Ca²⁺ sparks to total SR Ca²⁺ leak

A, relationships between SR Ca²⁺ leak rate and [Ca²⁺]_SR (n= 16 myocytes). Black circles and black line represent experimentally measured total leak. The leak data points between [Ca²⁺]_SR of 25 and 325 μm were fitted with a single sigmoid function and represents the non-spark-mediated leak (green line). Spark-mediated leak as function of [Ca²⁺]_SR was obtained by subtracting non-spark-mediated leak (green line) from total SR Ca²⁺ leak (black circles). The calculated points (red circles) could be fitted with a single exponential function (red line). B, dependence of total spark signal mass (black circles) and total spark-mediated Ca²⁺ release flux (red circles) from [Ca²⁺]_SR. Signal mass and Ca²⁺ release flux of all detected sparks were summated and normalized to the recording time (4.5 s). Spark signal mass and spark-mediated release flux were calculated as described in Methods. C, simultaneously recorded Ca²⁺ spark and blink. Top, line-scan image of Rhod-2 fluorescence and corresponding spark profile (F/F₀). Bottom, line-scan image of Fluo-5N fluorescence and corresponding blink profile. Spark and blink profiles were obtained by averaging fluorescence from the 1 μm the wide regions marked by the red and green box, respectively. D, distribution of [Ca²⁺]_SR at the nadir of blinks (44 events).

Figure 2. Effect of [Ca²⁺]_SR on Ca²⁺ spark properties

The dependence of spark frequency (A), spark amplitude (B), spark width (C) (measured at half-maximal amplitude, FWHM) and spark duration (D) (measured at half-maximal amplitude, FDHM) on [Ca²⁺]_SR (bin size 50 μm; n= 16 myocytes).

Figure 6. Components of SR Ca²⁺ leak and role of IP₃Rs in SR Ca²⁺ leak

A, different components of SR Ca²⁺ leak rate as a function of [Ca²⁺]_SR. Grey line represents the total RyR-mediated Ca²⁺ leak (spark and non-spark). This component was obtained by subtracting RuR-insensitive Ca²⁺ leak (green line) from the total Ca²⁺ leak (black line). Ca²⁺ spark-mediated leak (red line) was obtained as described in Fig. 3A. Non-spark RyR-mediated Ca²⁺ leak (blue line) was obtained by subtracting Ca²⁺ spark-mediated leak (red line) from the total RyR-mediated Ca²⁺ leak (grey line). B, the relationship between SR Ca²⁺ leak rate and [Ca²⁺]_SR in the presence of RuR (50 μm; black circles), in the presence of RuR plus 2-APB (20 μm; open circles) and in the presence of RuR plus IP₃ (10 μm; red circles).

Figure 5. Effects of RyR inhibitors on Ca²⁺ sparks, [Ca²⁺]_SR and SR Ca²⁺ leak

Effect of ruthenium red (RuR; 50 μm) (A) and Mg²⁺ (15 mm) (B) on spark frequency and [Ca²⁺]_SR before and after SERCA inhibition. For comparison, the dashed lines indicate the decline of [Ca²⁺]_SR in the absence of RyR inhibition (data from Fig. 1C). C, average effect of RuR (50 μm), Mg²⁺ (15 mm) and tetracaine (1 mm) on [Ca²⁺]_SR in the absence of TG. D, the relationships between SR Ca²⁺ leak rate and [Ca²⁺]_SR in control conditions (back), in the presence of RuR (green), tetracaine (blue) and Mg²⁺ (red). For presentation purposes only the fit to the data is shown for tetracaine and Mg²⁺.

Figure 7. Properties of SR Ca²⁺ leak in HF myocytes

A, changes of spark frequency and [Ca²⁺]_SR after SERCA inhibition with TG (10 μm) in myocyte from failing hearts. B, the total (circles) and RuR-insensitive (squares) SR Ca²⁺ leak as a function of [Ca²⁺]_SR in normal (black) and in HF myocytes (red). C, Ca²⁺ spark frequency in normal and HF myocytes measured at the same [Ca²⁺]_SR (680 μm).